Methods of producing 7-carbon chemicals via carbon chain elongation associated with cyclohexane carboxylate synthesis

ABSTRACT

This document describes biochemical pathways for producing pimelic acid, 7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine or 1,7-heptanediol by forming two terminal functional groups, comprised of carboxyl, amine or hydroxyl group, in a C7 aliphatic backbone substrate. These pathways, metabolic engineering and cultivation strategies described herein rely on the carbon chain elongation enzymes or homologs thereof associated with the cyclohexane carboxylate biosynthesis from  Syntrophus aciditrophicus  or 2-aminoadipate lysine biosynthesis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of U.S. Provisional Application Ser.No. 61/747,409, filed Dec. 31, 2012, and U.S. Provisional ApplicationSer. No. 61/829,092, filed May 30, 2013. The contents of the priorapplications are incorporated herein by reference in their entirety.

TECHNICAL FIELD

This invention relates to methods for biosynthesizing one or more ofpimelic acid, 7-hydroxyheptanoate, 7-aminoheptanoate,heptamethylenediamine and 1,7-heptanediol (hereafter “C7 buildingblocks”) using one or more isolated enzymes such as β-ketothiolases,dehydrogenases, reductases, thioesterases, decarboxylases, hydratases,synthases, thioesterases, CoA-ligases, CoA-transferases, deacetylases,or transaminases or using recombinant host cells expressing one or moresuch enzymes or using recombinant host cells expressing one or more suchenzymes.

BACKGROUND

Nylons are polyamides which are generally synthesized by thecondensation polymerisation of a diamine with a dicarboxylic acid.Similarly, Nylons may be produced by the condensation polymerisation oflactams. A ubiquitous Nylon is Nylon 6,6, which is produced by reactionof hexamethylenediamine (HMD) and adipic acid. Nylon 6 can be producedby a ring opening polymerisation of caprolactam (Anton & Baird,Polyamides Fibers, Encyclopedia of Polymer Science and Technology,2001).

Nylon 7 and Nylon 7,7 represent novel polyamides with value-addedcharacteristics compared to Nylon 6 and Nylon 6,6. Nylon 7 is producedby polymerisation of 7-aminoheptanoic acid, whereas Nylon 7,7 isproduced by condensation polymerisation of pimelic acid andheptamethylenediamine. No economically viable petrochemical routes existto producing the monomers for Nylon 7 and Nylon 7,7.

Given no economically viable petrochemical monomer feedstocks;biotechnology offers an alternative approach via biocatalysis.Biocatalysis is the use of biological catalysts, such as enzymes, toperform biochemical transformations of organic compounds.

Both bioderived feedstocks and petrochemical feedstocks are viablestarting materials for the biocatalysis processes.

Accordingly, against this background, it is clear that there is a needfor sustainable methods for producing one or more of pimelic acid,7-hydroxyheptanoate, 7-aminoheptanoate, heptamethylenediamine and1,7-heptanediol (hereafter “C7 building blocks”) wherein the methods arebiocatalyst based.

However, no wild-type prokaryote or eukaryote naturally overproduces orexcretes such C7 building blocks to the extracellular environment.Nevertheless, the metabolism of pimelic acid has been reported.

The dicarboxylic acid, pimelic acid, is converted efficiently as acarbon source by a number of bacteria and yeasts via β-oxidation intocentral metabolites. β-oxidation of Coenzyme A (CoA) activated pimelateto CoA activated 3-oxopimelate facilitates further catabolism via, forexample, pathways associated with aromatic substrate degradation. Thecatabolism of 3-oxopimeloyl-CoA to acetyl-CoA and glutaryl-CoA byseveral bacteria has been characterized comprehensively (Harwood andParales, Annual Review of Microbiology, 1996, 50:553-590).

The optimality principle states that microorganisms regulate theirbiochemical networks to support maximum biomass growth. Beyond the needfor expressing heterologous pathways in a host organism, directingcarbon flux towards C7 building blocks that serve as carbon sourcesrather than as biomass growth constituents, contradicts the optimalityprinciple. For example, transferring the 1-butanol pathway fromClostridium species into other production strains has often fallen shortby an order of magnitude compared to the production performance ofnative producers (Shen et al., Appl. Environ. Microbiol., 2011,77(9):2905-2915).

The efficient synthesis of the seven carbon aliphatic backbone precursoris a key consideration in synthesizing one or more C7 building blocksprior to forming terminal functional groups, such as carboxyl, amine orhydroxyl groups, on the C7 aliphatic backbone.

SUMMARY

This document is based at least in part on the discovery that it ispossible to construct biochemical pathways for producing a seven carbonchain aliphatic backbone precursor in which one or two functionalgroups, i.e., carboxyl, amine or hydroxyl, can be formed, leading to thesynthesis of one or more of pimelic acid, 7-aminoheptanoic acid,7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol(hereafter “C7 building blocks). Pimelic acid and pimilate,7-hydroxyheptanoic acid and 7-hydroxyheptanoate, and 7-aminoheptanoicand 7-aminoheptanoate are used interchangeably herein to refer to thecompound in any of its neutral or ionized forms, including any saltforms thereof. It is understood by those skilled in the art that thespecific form will depend on pH. These pathways, metabolic engineeringand cultivation strategies described herein rely on carbon chainelongation enzymes or homologs associated with cyclohexane carboxylatebiosynthesis in Synthrophus aciditrophicus or 2-aminoadipate lysinebiosynthesis.

In the face of the optimality principle, it surprisingly has beendiscovered that appropriate non-natural pathways, feedstocks, hostmicroorganisms, attenuation strategies to the host's biochemicalnetwork, and cultivation strategies may be combined to efficientlyproduce one or more C7 building blocks.

In some embodiments, the C7 aliphatic backbone for conversion to a C7building block is 3-ketopimeloyl-CoA (also can be referred to as3-oxopimeloyl-CoA) or pimeloyl-CoA, which can be formed from acetyl-CoAusing enzymes associated with cyclohexane carboxylate biosynthesis inSynthrophus aciditrophicus or 2-aminoadipate lysine biosynthesis. SeeFIG. 1 and FIG. 2.

In some embodiments, a terminal carboxyl group can be enzymaticallyformed using a thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a CoA-transferase, or a reversibleCoA-ligase. See FIG. 1, FIG. 2 and FIG. 3.

In some embodiments, a terminal amine group can be enzymatically formedusing a ω-transaminase or a deacetylase. See FIG. 4 and FIG. 5.

In some embodiments, a terminal hydroxyl group can be enzymaticallyformed using an alcohol dehydrogenase. See FIG. 6 and FIGS. 7A-7H.

In one aspect, this document features a method for biosynthesizing aproduct selected from the group consisting of pimelic acid,7-aminoheptanoic acid, 7-hydroxyheptanoic acid, heptamethylenediamine,and 1,7-heptanediol. The method includes enzymatically synthesizing aseven carbon chain aliphatic backbone using enzymes associated withcyclohexane carboxylate biosynthesis or the 2-aminoadipate lysinebiosynthesis pathway, and enzymatically forming one or two terminalfunctional groups selected from the group consisting of carboxyl, amine,and hydroxyl groups in the backbone, thereby forming the product. Theseven carbon chain aliphatic backbone can be pimeloyl-CoA. Pimeloyl-CoAcan be enzymatically synthesized from acetyl-CoA or 2-oxoglutarate via3-ketopimeloyl-CoA. A β-ketothiolase or a β-ketoacyl [acp] synthase canconvert glutaryl-CoA to 3-ketopimeloyl-CoA. Glutaryl-CoA can be producedfrom crotonyl-CoA using a glutaconyl-CoA decarboxylase or a glutaryl-CoAdehydrogenase. 3-ketopimeloyl-CoA can be converted to3-hydroxypimeloyl-CoA using a 3-hydroxyadipyl-CoA dehydrogenase;3-hydroxypimeloyl-CoA can be converted to6-ketocyclohex-1-ene-1-carboxyl-CoA by a6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or 3-hydroxypimeloyl-CoA canbe converted to 2,3-dehydropimeloyl-CoA by an enoyl-CoA hydratase.

The two terminal functional groups can be the same (e.g., amine orhydroxyl) or can be different (e.g., a terminal amine and a terminalcarboxyl group; or a terminal hydroxyl group and a terminal carboxylgroup).

A ω-transaminase or a deacetylase can enzymatically form an amine group.The ω-transaminase can have at least 70% sequence identity to any one ofthe amino acid sequences set forth in SEQ ID NO. 8-13.

A 6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase,a 4-hydroxybutyrate dehydratase, or an alcohol dehydrogenase canenzymatically form a hydroxyl group.

A thioesterase, an aldehyde dehydrogenase, a 7-oxoheptanoatedehydrogenase, a 6-oxohexanoate dehydrogenase, a CoA-transferase (e.g. aglutaconate CoA transferase), or a reversible CoA-ligase (e.g., areversible succinate-CoA ligase) can enzymatically forms a terminalcarboxyl group. The thioesterase can have at least 70% sequence identityto the amino acid sequence set forth in SEQ ID NO: 1.

A carboxylate reductase and a phosphopantetheinyl transferase can form aterminal aldehyde group as an intermediate in forming the product. Thecarboxylate reductase can have at least 70% sequence identity to any oneof the amino acid sequences set forth in SEQ ID NO. 2-7.

Any of the methods can be performed in a recombinant host byfermentation. The host can be subjected to a cultivation strategy underaerobic, anaerobic, micro-aerobic or mixed oxygen/denitrificationcultivation conditions. The host can be cultured under conditions ofnutrient limitation. The host can be retained using a ceramic hollowfiber membrane to maintain a high cell density during fermentation.

In any of the methods, the host's tolerance to high concentrations of aC7 building block can be improved through continuous cultivation in aselective environment.

The principal carbon source fed to the fermentation can derive frombiological or non-biological feedstocks. In some embodiments, thebiological feedstock is, includes, or derives from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid and formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste.

In some embodiments, the non-biological feedstock is or derives fromnatural gas, syngas, CO₂/H₂, methanol, ethanol, benzoate, non-volatileresidue (NVR) or a caustic wash waste stream from cyclohexane oxidationprocesses, or a terephthalic acid/isophthalic acid mixture waste stream.

In another aspect, this document features a recombinant host thatincludes at least one exogenous nucleic acid encoding i) aβ-ketothiolase or a β-ketoacyl[acp] synthase, (ii) a6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA hydratase,and (iii) a trans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a2-ketocyclohexanecarboxyl-CoA hydrolase, the host producingpimeloyl-CoA. The host further can include a glutaconyl-CoAdecarboxylase or a glutaryl-CoA dehydrogenase.

A recombinant host producing pimeloyl-CoA further can include at leastone exogenous nucleic acid encoding one or more of a thioesterase, analdehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a6-oxohexanoate dehydrogenase, a glutaconate CoA-transferase, areversible succinyl-CoA ligase, an acetylating aldehyde dehydrogenase,or a carboxylate reductase, the host producing pimelic acid or pimelatesemialdehyde.

A recombinant host producing pimelate semialdehyde further can includeat least one exogenous nucleic acid encoding a ω-transaminase, andproducing 7-aminoheptanoate.

A recombinant host producing pimelate semialdehyde further can includeat least one exogenous nucleic acid encoding a 4-hydroxybutyratedehydrogenase, a 5-hydroxypentanoate dehydrogenase or a6-hydroxyhexanoate dehydrogenase, the host producing 7-hydroxyheptanoicacid.

A recombinant host producing pimelate semialdehyde, 7-aminoheptanoate,or 7-hydroxyheptanoic acid further can include a carboxylate reductase,a ω-transaminase, a deacetylase, an N-acetyl transferase, or an alcoholdehydrogenase, the host producing heptamethylenediamine.

A recombinant host producing 7-hydroxyheptanoic acid further can includeat least one exogenous nucleic acid encoding a carboxylate reductase oran alcohol dehydrogenase, the host producing 1,7-heptanediol.

The recombinant host can be a prokaryote, e.g., from the genusEscherichia such as Escherichia coli; from the genus Clostridia such asClostridium ljungdahlii, Clostridium autoethanogenum or Clostridiumkluyveri; from the genus Corynebacteria such as Corynebacteriumglutamicum; from the genus Cupriavidus such as Cupriavidus necator orCupriavidus metallidurans; from the genus Pseudomonas such asPseudomonas fluorescens, Pseudomonas putida or Pseudomonas oleavorans;from the genus Delftia acidovorans, from the genus Bacillus such asBacillus subtillis; from the genes Lactobacillus such as Lactobacillusdelbrueckii; from the genus Lactococcus such as Lactococcus lactis orfrom the genus Rhodococcus such as Rhodococcus equi.

The recombinant host can be a eukaryote, e.g., a eukaryote from thegenus Aspergillus such as Aspergillus niger; from the genusSaccharomyces such as Saccharomyces cerevisiae; from the genus Pichiasuch as Pichia pastoris; from the genus Yarrowia such as Yarrowialipolytica, from the genus Issatchenkia such as Issathenkia orientalis,from the genus Debaryomyces such as Debaryomyces hansenii, from thegenus Arxula such as Arxula adenoinivorans, or from the genusKluyveromyces such as Kluyveromyces lactis.

Any of the recombinant hosts described herein further can include one ormore of the following attenuated enzymes: a phosphotransacetylasegenerating acetate, an acetate kinase; a lactate dehydrogenase; amenaquinol-fumarate oxidoreductase; an alcohol dehydrogenase producingethanol; a pyruvate decarboxylase; a 2-oxoacid decarboxylase generatingisobutanol; a polymer synthase; a NADPH-specific L-glutamatedehydrogenase; a NADPH/NADH L-glutamate dehydrogenase; a NADH-consumingtranshydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoAdehydrogenase that degrades C7 building blocks and their precursors; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase.

Any of the recombinant hosts described herein further can overexpressone or more genes encoding: an acetyl-CoA synthetase, a formatedehydrogenase, a PEP carboxykinase, a PEP carboxylase, a pyruvatecarboxylase, a PEP synthase, a L-alanine dehydrogenase; a NADH-specificL-glutamate dehydrogenase; a diamine transporter; a dicarboxylatetransporter; and/or a multidrug transporter.

The reactions of the pathways described herein can be performed in oneor more cell (e.g., host cell) strains (a) naturally expressing one ormore relevant enzymes, (b) genetically engineered to express one or morerelevant enzymes, or (c) naturally expressing one or more relevantenzymes and genetically engineered to express one or more relevantenzymes. Alternatively, relevant enzymes can be extracted from any ofthe above types of host cells and used in a purified or semi-purifiedform. Extracted enzymes can optionally be immobilized on a solidsubstrate such as the floors and/or walls of appropriate reactionvessels. Moreover, such extracts include lysates (e.g. cell lysates)that can be used as sources of relevant enzymes. In the methods providedby the document, all the steps can be performed in cells (e.g., hostcells), all the steps can be performed using extracted enzymes, or someof the steps can be performed in cells and others can be performed usingextracted enzymes.

Many of the enzymes described herein catalyze reversible reactions, andthe reaction of interest may be the reverse of the described reaction.The schematic pathways shown in FIGS. 1-6 illustrate the reaction ofinterest for each of the intermediates.

In some embodiments, the host microorganism's endogenous biochemicalnetwork is attenuated or augmented to (1) ensure the intracellularavailability of acetyl-CoA (2) create an NADH imbalance that may only bebalanced via the formation of a C7 building block, (3) preventdegradation of central metabolites or central precursors leading to andincluding C7 building blocks and (4) ensure efficient efflux from thecell.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and the drawings, and from the claims. The word “comprising”in the claims may be replaced by “consisting essentially of” or with“consisting of,” according to standard practice in patent law.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic of an exemplary biochemical pathway leading topimeloyl-CoA from acetyl-CoA or 2-oxo-glutarate as a central metabolite.

FIG. 2 is a schematic of exemplary biochemical pathways leading topimelate using pimeloyl-CoA as a central precursor.

FIG. 3 is a schematic of exemplary biochemical pathways leading to7-aminoheptanoate using pimeloyl-CoA or pimelate as a central precursor.

FIG. 4 is a schematic of an exemplary biochemical pathway leading toheptamethylenediamine using 7-aminheptanoate, 7-hydroxyheptanoate, orpimelate semialdehyde as a central precursor.

FIG. 5 is a schematic of an exemplary biochemical pathway leading to7-hydroxyheptanoate using pimeloyl-CoA or pimelic acid as a centralprecursor.

FIG. 6 is a schematic of an exemplary biochemical pathway leading to1,7-heptanediol using 7-hydroxyheptanoate as a central precursor.

FIGS. 7A-7H contain the amino acid sequences of an Escherichia colithioesterase encoded by tesB (see GenBank Accession No. AAA24665.1, SEQID NO: 1), a Mycobacterium marinum carboxylate reductase (see GenbankAccession No. ACC40567.1, SEQ ID NO: 2), a Mycobacterium smegmatiscarboxylate reductase (see Genbank Accession No. ABK71854.1, SEQ ID NO:3), a Segniliparus rugosus carboxylate reductase (see Genbank AccessionNo. EFV11917.1, SEQ ID NO: 4), a Mycobacterium smegmatis carboxylatereductase (see Genbank Accession No. ABK75684.1, SEQ ID NO: 5), aMycobacterium massiliense carboxylate reductase (see Genbank AccessionNo. EIV11143.1, SEQ ID NO: 6), a Segniliparus rotundus carboxylatereductase (see Genbank Accession No. ADG98140.1, SEQ ID NO: 7), aChromobacterium violaceum ω-transaminase (see Genbank Accession No.AAQ59697.1, SEQ ID NO: 8), a Pseudomonas aeruginosa ω-transaminase (seeGenbank Accession No. AAG08191.1, SEQ ID NO: 9), a Pseudomonas syringaeω-transaminase (see Genbank Accession No. AAY39893.1, SEQ ID NO: 10), aRhodobacter sphaeroides ω-transaminase (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli ω-transaminase (seeGenbank Accession No. AAA57874.1, SEQ ID NO: 12), a Vibrio fluvialisω-transaminase (see Genbank Accession No. AEA39183.1, SEQ ID NO: 13), aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858.1, SEQ ID NO:14), or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:15).

FIG. 8 is a bar graph of the relative absorbance at 412 nm of releasedCoA as a measure of the activity of a thioesterase for convertingpimeloyl-CoA to pimelate relative to the empty vector control.

FIG. 9 is a bar graph summarizing the change in absorbance at 340 nmafter 20 minutes, which is a measure of the consumption of NADPH andactivity of carboxylate reductases relative to the enzyme only controls(no substrate).

FIG. 10 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting pimelate to pimelatesemialdehyde relative to the empty vector control.

FIG. 11 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting 7-hydroxyheptanoate to7-hydroxyheptanal relative to the empty vector control.

FIG. 12 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and the activityof carboxylate reductases for converting N7-acetyl-7-aminoheptanoate toN7-acetyl-7-aminoheptanal relative to the empty vector control.

FIG. 13 is a bar graph of the change in absorbance at 340 nm after 20minutes, which is a measure of the consumption of NADPH and activity ofcarboxylate reductases for converting pimelate semialdehyde toheptanedial relative to the empty vector control.

FIG. 14 is a bar graph summarizing the percent conversion after 4 hoursof pyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity of the enzyme only controls (no substrate) or the empty vector.

FIG. 15 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 7-aminoheptanoate to pimelate semialdehyderelative to the empty vector control.

FIG. 16 is a bar graph of the percent conversion after 4 hours ofL-alanine to pyruvate (mol/mol) as a measure of the ω-transaminaseactivity for converting pimelate semialdehyde to 7-aminoheptanoaterelative to the empty vector control.

FIG. 17 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting heptamethylene diamine to 7-aminoheptanalrelative to the empty vector control.

FIG. 18 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting N7-acetyl-1,7-diaminoheptane toN7-acetyl-7-aminoheptanal relative to the empty vector control.

FIG. 19 is a bar graph of the percent conversion after 4 hours ofpyruvate to L-alanine (mol/mol) as a measure of the ω-transaminaseactivity for converting 7-aminoheptanol to 7-hydroxyheptanal relative tothe empty vector control.

DETAILED DESCRIPTION

This document provides enzymes, non-natural pathways, cultivationstrategies, feedstocks, host microorganisms and attenuations to thehost's biochemical network, which generates a seven carbon chainaliphatic backbone from central metabolites in which one or two terminalfunctional groups may be formed leading to the synthesis of one or moreof pimelic acid, 7-hydroxyheptanoic acid, 7-aminoheptanoic acid,heptamethylenediamine, and 1,7-heptanediol (referred to as “C7 buildingblocks” herein). As used herein, the term “central precursor” is used todenote any metabolite in any metabolic pathway shown herein leading tothe synthesis of a C7 building block. The term “central metabolite” isused herein to denote a metabolite that is produced in allmicroorganisms to support growth.

Host microorganisms described herein can include endogenous pathwaysthat can be manipulated such that one or more C7 building blocks can beproduced. In an endogenous pathway, the host microorganism naturallyexpresses all of the enzymes catalyzing the reactions within thepathway. A host microorganism containing an engineered pathway does notnaturally express all of the enzymes catalyzing the reactions within thepathway but has been engineered such that all of the enzymes within thepathway are expressed in the host.

The term “exogenous” as used herein with reference to a nucleic acid (ora protein) and a host refers to a nucleic acid that does not occur in(and cannot be obtained from) a cell of that particular type as it isfound in nature or a protein encoded by such a nucleic acid. Thus, anon-naturally-occurring nucleic acid is considered to be exogenous to ahost once in the host. It is important to note thatnon-naturally-occurring nucleic acids can contain nucleic acidsubsequences or fragments of nucleic acid sequences that are found innature provided the nucleic acid as a whole does not exist in nature.For example, a nucleic acid molecule containing a genomic DNA sequencewithin an expression vector is non-naturally-occurring nucleic acid, andthus is exogenous to a host cell once introduced into the host, sincethat nucleic acid molecule as a whole (genomic DNA plus vector DNA) doesnot exist in nature. Thus, any vector, autonomously replicating plasmid,or virus (e.g., retrovirus, adenovirus, or herpes virus) that as a wholedoes not exist in nature is considered to be non-naturally-occurringnucleic acid. It follows that genomic DNA fragments produced by PCR orrestriction endonuclease treatment as well as cDNAs are considered to benon-naturally-occurring nucleic acid since they exist as separatemolecules not found in nature. It also follows that any nucleic acidcontaining a promoter sequence and polypeptide-encoding sequence (e.g.,cDNA or genomic DNA) in an arrangement not found in nature isnon-naturally-occurring nucleic acid. A nucleic acid that isnaturally-occurring can be exogenous to a particular host microorganism.For example, an entire chromosome isolated from a cell of yeast x is anexogenous nucleic acid with respect to a cell of yeast y once thatchromosome is introduced into a cell of yeast y.

In contrast, the term “endogenous” as used herein with reference to anucleic acid (e.g., a gene) (or a protein) and a host refers to anucleic acid (or protein) that does occur in (and can be obtained from)that particular host as it is found in nature. Moreover, a cell“endogenously expressing” a nucleic acid (or protein) expresses thatnucleic acid (or protein) as does a host of the same particular type asit is found in nature. Moreover, a host “endogenously producing” or that“endogenously produces” a nucleic acid, protein, or other compoundproduces that nucleic acid, protein, or compound as does a host of thesame particular type as it is found in nature.

For example, depending on the host and the compounds produced by thehost, one or more of the following enzymes may be expressed in the hostin addition to (i) a β-ketothiolase or a β-ketoacyl[acp] synthase; (ii)a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase or an enoyl-CoA hydrataseaccepting 3-hydroxypimeloyl-CoA as substrate, and (iii) atrans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a2-ketocyclohexanecarboxyl-CoA hydrolase: a glutaconyl-CoA decarboxylase,a glutaryl-CoA dehydrogenase, a 3-hydroxyacyl-CoA dehydrogenase, a6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase, acyclohexa-1,5-dienecarbonyl-CoA hydratase, acyclohex-1-ene-1-carboxyl-CoA dehydrogenase, acyclohex-1-ene-1-carboxyl-CoA hydratase, a2-hydroxycyclohexanecarboxyl-CoA dehydrogenase, a thioesterase, analdehyde dehydrogenase, a 7-oxoheptanoate dehydrogenase, a6-oxohexanoate dehydrogenase, a 4-hydroxybutyrate dehydrogenase, a5-hydroxypentanoate dehydrogenase, a 6-hydroxyhexanoate dehydrogenase, aCoA-transferase, a reversible CoA-ligase, an acetylating aldehydedehydrogenase, a ω-transaminase, a carboxylate reductase, a deacetylase,a lysine N-acetyl transferase, or an alcohol dehydrogenase. Inrecombinant hosts expressing a carboxylate reductase, aphosphopantetheinyl transferase also can be expressed as it enhancesactivity of the carboxylate reductase.

For example, a recombinant host can include a β-ketothiolase or aβ-ketoacyl[acp] synthase; a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolaseor an enoyl-CoA hydratase accepting 3-hydroxypimeloyl-CoA as substrate;and a trans-2-enoyl-CoA reductase, an enoyl-[acp] reductase, or a2-ketocyclohexanecarboxyl-CoA hydrolase, and produce pimeloyl-CoA. Thehost further can include a glutaconyl-CoA decarboxylase or aglutaryl-CoA dehydrogenase.

A recombinant host producing pimeloyl-CoA further can include one ormore of an aldehyde dehydrogenase, a 6-oxohexanoate dehydrogenase, athioesterase, a 7-oxoheptanoate dehydrogenase, a reversiblesuccinyl-CoA-ligase, a glutaconate CoA transferase, a carboxylatereductase, or an acetylating aldehyde dehydrogenase, and producepimelate or pimelate semialdehyde. For example, a recombinant hostproducing pimeloyl-CoA can include a thioesterase, a CoA-transferase, ora reversible CoA-ligase and produce pimelic acid. For example, arecombinant host producing pimeloyl-CoA can include an acetylatingaldehyde dehydrogenase and either an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase andproduce pimelic acid. For example, a recombinant host producingpimeloyl-CoA can include an acetylating aldehyde dehydrogenase or acarboxylate reductase and produce pimelate semialdehyde.

A recombinant host producing pimelic acid or pimelate semialdehydefurther can include at least one exogenous nucleic acid encoding aω-transaminase and produce 7-aminoheptanoate.

A recombinant host producing pimelate semialdehyde further can include a6-hydroxyhexanoate dehydrogenase, a 5-hydroxypentanoate dehydrogenase ora 4-hydroxybutyrate dehydrogenase, and produce 7-hydroxyheptanoic acid.

A recombinant host producing pimelate semialdehyde, 7-aminoheptanoate,or 7-hydroxyheptanoate further can include at least one exogenousnucleic acid encoding a carboxylate reductase, a ω-transaminase, adeacetylase, a lysine N-acetyl transferase, or an alcohol dehydrogenase,and produce heptamethylenediamine. For example, a recombinant hostproducing 7-hydroxyheptanoate can include a carboxylate reductase, aω-transaminase and an alcohol dehydrogenase.

A recombinant host producing 7-hydroxyheptanoic acid further can includea carboxylate reductase or an alcohol dehydrogenase, and produce1,7-heptanediol.

Within an engineered pathway, the enzymes can be from a single source,i.e., from one species or genus, or can be from multiple sources, i.e.,different species or genera. Nucleic acids encoding the enzymesdescribed herein have been identified from various organisms and arereadily available in publicly available databases such as GenBank orEMBL.

Any of the enzymes described herein that can be used for production ofone or more C7 building blocks can have at least 70% sequence identity(homology) (e.g., at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100%) to the amino acid sequence of the corresponding wild-type enzyme.It will be appreciated that the sequence identity can be determined onthe basis of the mature enzyme (e.g., with any signal sequence removed).

For example, a thioesterase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of an Escherichiacoli thioesterase encoded by tesB (see GenBank Accession No. AAA24665.1,SEQ ID NO: 1). See FIG. 7A.

For example, a carboxylate reductase described herein can have at least70% sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%,95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aMycobacterium marinum (see Genbank Accession No. ACC40567.1, SEQ ID NO:2), a Mycobacterium smegmatis (see Genbank Accession No. ABK71854.1, SEQID NO: 3), a Segniliparus rugosus (see Genbank Accession No. EFV11917.1,SEQ ID NO: 4), a Mycobacterium smegmatis (see Genbank Accession No.ABK75684.1, SEQ ID NO: 5), a Mycobacterium massiliense (see GenbankAccession No. EIV11143.1, SEQ ID NO: 6), or a Segniliparus rotundus (seeGenbank Accession No. ADG98140.1, SEQ ID NO: 7) carboxylate reductase.See, FIGS. 7A-7F.

For example, a ω-transaminase described herein can have at least 70%sequence identity (homology) (e.g., at least 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100%) to the amino acid sequence of a Chromobacteriumviolaceum (see Genbank Accession No. AAQ59697.1, SEQ ID NO: 8), aPseudomonas aeruginosa (see Genbank Accession No. AAG08191.1, SEQ ID NO:9), a Pseudomonas syringae (see Genbank Accession No. AAY39893.1, SEQ IDNO: 10), a Rhodobacter sphaeroides (see Genbank Accession No.ABA81135.1, SEQ ID NO: 11), an Escherichia coli (see Genbank AccessionNo. AAA57874.1, SEQ ID NO: 12), or a Vibrio fluvialis (see GenbankAccession No. AEA39183.1, SEQ ID NO: 13) ω-transaminase. Some of theseω-transaminases are diamine ω-transaminases. See, FIGS. 7F and 7G.

For example, a phosphopantetheinyl transferase described herein can haveat least 70% sequence identity (homology) (e.g., at least 75%, 80%, 85%,90%, 95%, 97%, 98%, 99%, or 100%) to the amino acid sequence of aBacillus subtilis phosphopantetheinyl transferase (see Genbank AccessionNo. CAA44858.1, SEQ ID NO:14) or a Nocardia sp. NRRL 5646phosphopantetheinyl transferase (see Genbank Accession No. ABI83656.1,SEQ ID NO:15). See FIGS. 7G and 7H.

The percent identity (homology) between two amino acid sequences can bedetermined as follows. First, the amino acid sequences are aligned usingthe BLAST 2 Sequences (Bl2seq) program from the stand-alone version ofBLASTZ containing BLASTP version 2.0.14. This stand-alone version ofBLASTZ can be obtained from Fish & Richardson's web site (e.g.,www.fr.com/blast/) or the U.S. government's National Center forBiotechnology Information web site (www.ncbi.nlm.nih.gov). Instructionsexplaining how to use the Bl2seq program can be found in the readme fileaccompanying BLASTZ. Bl2seq performs a comparison between two amino acidsequences using the BLASTP algorithm. To compare two amino acidsequences, the options of Bl2seq are set as follows: -i is set to a filecontaining the first amino acid sequence to be compared (e.g.,C:\seq1.txt); -j is set to a file containing the second amino acidsequence to be compared (e.g., C:\seq2.txt); -p is set to blastp; -o isset to any desired file name (e.g., C:\output.txt); and all otheroptions are left at their default setting. For example, the followingcommand can be used to generate an output file containing a comparisonbetween two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -jc:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequencesshare homology (identity), then the designated output file will presentthose regions of homology as aligned sequences. If the two comparedsequences do not share homology (identity), then the designated outputfile will not present aligned sequences. Similar procedures can befollowing for nucleic acid sequences except that blastn is used.

Once aligned, the number of matches is determined by counting the numberof positions where an identical amino acid residue is presented in bothsequences. The percent identity (homology) is determined by dividing thenumber of matches by the length of the full-length polypeptide aminoacid sequence followed by multiplying the resulting value by 100. It isnoted that the percent identity (homology) value is rounded to thenearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 is roundeddown to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 is rounded upto 78.2. It also is noted that the length value will always be aninteger.

It will be appreciated that a number of nucleic acids can encode apolypeptide having a particular amino acid sequence. The degeneracy ofthe genetic code is well known to the art; i.e., for many amino acids,there is more than one nucleotide triplet that serves as the codon forthe amino acid. For example, codons in the coding sequence for a givenenzyme can be modified such that optimal expression in a particularspecies (e.g., bacteria or fungus) is obtained, using appropriate codonbias tables for that species.

Functional fragments of any of the enzymes described herein can also beused in the methods of the document. The term “functional fragment” asused herein refers to a peptide fragment of a protein that has at least25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%; 90%; 95%;98%; 99%; 100%; or even greater than 100%) of the activity of thecorresponding mature, full-length, wild-type protein. The functionalfragment can generally, but not always, be comprised of a continuousregion of the protein, wherein the region has functional activity.

This document also provides (i) functional variants of the enzymes usedin the methods of the document and (ii) functional variants of thefunctional fragments described above. Functional variants of the enzymesand functional fragments can contain additions, deletions, orsubstitutions relative to the corresponding wild-type sequences. Enzymeswith substitutions will generally have not more than 50 (e.g., not morethan one, two, three, four, five, six, seven, eight, nine, ten, 12, 15,20, 25, 30, 35, 40, or 50) amino acid substitutions (e.g., conservativesubstitutions). This applies to any of the enzymes described herein andfunctional fragments. A conservative substitution is a substitution ofone amino acid for another with similar characteristics. Conservativesubstitutions include substitutions within the following groups: valine,alanine and glycine; leucine, valine, and isoleucine; aspartic acid andglutamic acid; asparagine and glutamine; serine, cysteine, andthreonine; lysine and arginine; and phenylalanine and tyrosine. Thenonpolar hydrophobic amino acids include alanine, leucine, isoleucine,valine, proline, phenylalanine, tryptophan and methionine. The polarneutral amino acids include glycine, serine, threonine, cysteine,tyrosine, asparagine and glutamine. The positively charged (basic) aminoacids include arginine, lysine and histidine. The negatively charged(acidic) amino acids include aspartic acid and glutamic acid. Anysubstitution of one member of the above-mentioned polar, basic or acidicgroups by another member of the same group can be deemed a conservativesubstitution. By contrast, a nonconservative substitution is asubstitution of one amino acid for another with dissimilarcharacteristics.

Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. Additions (addition variants) include fusion proteins containing:(a) any of the enzymes described herein or a fragment thereof; and (b)internal or terminal (C or N) irrelevant or heterologous amino acidsequences. In the context of such fusion proteins, the term“heterologous amino acid sequences” refers to an amino acid sequenceother than (a). A heterologous sequence can be, for example a sequenceused for purification of the recombinant protein (e.g., FLAG,polyhistidine (e.g., hexahistidine), hemagglutinin (HA),glutathione-S-transferase (GST), or maltosebinding protein (MBP)).Heterologous sequences also can be proteins useful as detectablemarkers, for example, luciferase, green fluorescent protein (GFP), orchloramphenicol acetyl transferase (CAT). In some embodiments, thefusion protein contains a signal sequence from another protein. Incertain host cells (e.g., yeast host cells), expression and/or secretionof the target protein can be increased through use of a heterologoussignal sequence. In some embodiments, the fusion protein can contain acarrier (e.g., KLH) useful, e.g., in eliciting an immune response forantibody generation) or ER or Golgi apparatus retention signals.Heterologous sequences can be of varying length and in some cases can bea longer sequences than the full-length target proteins to which theheterologous sequences are attached.

Engineered hosts can naturally express none or some (e.g., one or more,two or more, three or more, four or more, five or more, or six or more)of the enzymes of the pathways described herein. Thus, a pathway withinan engineered host can include all exogenous enzymes, or can includeboth endogenous and exogenous enzymes. Endogenous genes of theengineered hosts also can be disrupted to prevent the formation ofundesirable metabolites or prevent the loss of intermediates in thepathway through other enzymes acting on such intermediates. Engineeredhosts can be referred to as recombinant hosts or recombinant host cells.As described herein recombinant hosts can include nucleic acids encodingone or more of a dehydrogenase, a β-ketothiolase, a β-ketoacyl-[acp]synthase, a reductase, a hydratase, a thioesterase, a CoA-ligase, areversible CoA-ligase, a CoA-transferase, an N-acetyl transferase, adeacetylase, or a ω-transaminase as described in detail herein.

In addition, the production of one or more C7 building blocks can beperformed in vitro using the isolated enzymes described herein, using alysate (e.g., a cell lysate) from a host microorganism as a source ofthe enzymes, or using a plurality of lysates from different hostmicroorganisms as the source of the enzymes.

Enzymes Generating the C7 Aliphatic Backbone, Pimeloyl-CoA, forConversion to C7 Building Blocks

As depicted in FIG. 1, pimeloyl-CoA, a C7 aliphatic backbone forconversion to a C7 building block, can be formed from the centralmetabolites acetyl-CoA or 2-oxoglutarate via carbon chain elongation (i)associated with cyclohexane carboxylate biosynthesis in Synthrophusaciditrophicus or (ii) associated with lysine biosynthesis viaα-aminoadipate.

In some embodiments, glutaryl-CoA is formed via CoA-dependent carbonchain elongation associated with cyclohexane carboxylate biosynthesis inSynthrophus aciditrophicus which comprises using (i) a β-ketothiolase oran acetyl-carboxylase in combination with an acetoacetyl-CoA synthase,(ii) a 3-hydroxybutyryl-CoA dehydrogenase, (iii) an enoyl-CoA hydratase,and either (iv) a glutaryl-CoA dehydrogenase in combination with anenoyl-CoA reductase or a trans-2-enoyl-CoA reductase or (v) aglutaconyl-CoA decarboxylase.

In some embodiments, glutaryl-CoA is formed via C1 carbon chainelongation associated with lysine biosynthesis via α-aminoadipate, whichcomprises using (i) a homocitrate synthase, (ii) a homocitratedehydratase and a homoaconitate hydratase, (iii) an isohomocitratedehydrogenase, (iv) an indolepyruvate decarboxylase, (vi) aglutarate-semialdehyde dehydrogenase and (v) a glutarate: CoA ligase.

In some embodiments, pimeloyl-CoA is formed from glutaryl-CoA via CoAdependent chain elongation using (i) a β-ketothiolase or β-ketoacyl[acp]synthase, (ii) a 3-hydroxyacyl-CoA dehydrogenase, (iii) an enoyl-CoAhydratase, and (iv) an enoyl-CoA reductase.

In some embodiments, pimeloyl-CoA is formed from 3-hydroxypimeloyl-CoAvia aromatic intermediates associated with cyclohexane carboxylatebiosynthesis in Synthrophus aciditrophicus using (i) a6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase, (ii) a6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase, (iii) acyclohexa-1,5-dienecarbonyl-CoA hydratase, (iv) acyclohex-1-ene-1-carboxyl-CoA dehydrogenase, (v) acyclohex-1-ene-1-carboxyl-CoA hydratase, (vi) a2-hydroxycyclohexanecarboxyl-CoA dehydrogenase and (vi) a2-ketocyclohexanecarboxyl-CoA hydrolase.

In some embodiments, a β-ketothiolase may be classified under EC 2.3.1.-(e.g., EC 2.3.1.9, EC 2.3.1.16, or EC 2.3.174). For example, aβ-ketothiolase may be classified under EC 2.3.1.9, such as the geneproduct of atoB or phaA. The β-ketothiolase encoded by atoB or phaAaccepts acetyl-CoA as substrates, forming butanoyl-CoA (see, Haywood etal., 1988, supra; Slater et al., 1998, supra). The β-ketothiolaseencoded by paaJ can be classified under, for example, EC 2.3.1.174. Theβ-ketothiolase encoded by paaJ condenses acetyl-CoA and succinyl-CoA to3-oxoadipyl-CoA (see, for example, Fuchs et al., 2011, Nature ReviewsMicrobiology, 9, 803-816). A homologue of paaJ in Synthrophusaciditrophicus catalyses the condensation of acetyl-CoA and glutaryl-CoAto 3-oxopimeloyl-CoA such as Genbank Accession No. ABC78517.1 or GenbankAccession No. ABC78881.1. Alternately, a β-ketoacyl[acp] homologue ofpaaJ in S. aciditrophicus catalyses the condensation of acetyl-CoA andglutaryl-CoA to 3-oxopimeloyl-CoA.

A β-ketoacyl[acp] synthase can be classified, for example, under, EC2.3.1.41, EC 2.3.1.179, or EC 2.3.1.180. The β-ketothiolases andβ-ketoacyl-[acp] synthases involved in fatty acid synthesis in S.aciditrophicus likely accept CoA activated dicarboxylic acids (Mouttakiet al., Appl. Environ. Microbiol., 2007, 73(3), 930-938).

An acetyl-CoA carboxylase can be classified under EC 6.4.1.2 and anacetoacetyl-CoA synthase can be classified under EC 2.3.1.194.Conversion of acetyl-CoA to malonyl-CoA by an acetyl-CoA carboxylase hasbeen shown to increase the rate of fatty acid synthesis (Davis et al.,J. Biol. Chem., 2000, 275(37), 28593-28598). It has been demonstratedthat acetoacetyl-CoA synthase may be used as an irreversible substitutefor the gene product of phaA in the carbon chain elongation associatedwith polyhydroxybutyrate synthesis (Matsumoto et al., Biosci.Biotechnol. Biochem., 2011, 75(2), 364-366).

In some embodiments, a 3-hydroxybutyryl-CoA dehydrogenase (also can bereferred to as a 3-hydroxyacyl-CoA dehydrogenase) may be classifiedunder EC 1.1.1.157 such as the gene product hbd (see, for example, Shenet al., 2011, supra; Budde et al., J. Bacteriol., 2010, 192(20),5319-5328).

In some embodiments, an enoyl-CoA hydratase can be classified under EC4.2.1.17, such as the gene product of crt (see, for example, Shen etal., 2011, supra; Fukui et al., J. Bacteriol., 1998, 180(3), 667-673) orthe gene product of paaF (see, for example, Fuchs et al., 2011, supra).Homologs of paaF in S. aciditrophicus include the enoyl-CoA hydratase ofGenbank Accession No. ABC77794.1 or the enoyl-CoA dehydratase of GenbankAccession No. ABC78950.1.

In some embodiments, an enoyl-[acp] reductase can be classified under EC1.3.1.- (e.g., EC 1.3.1.9) such as the enoyl-[acp] reductase obtainedfrom S. aciditrophicus or the gene product of FabI (Genbank AccessionNo: CAB13029.2) from Bacillus subtillis (see, for example, Heath et al.,2000, J. Biol. Chem., 275(51), 40128-33). The enoyl-[acp] reductaseinvolved in fatty acid synthesis in S. aciditrophicus likely accepts CoAactivated dicarboxylic acids (Mouttaki et al., 2007, supra).

In some embodiments, a trans-2-enoyl-CoA reductase can be classified,for example, under EC 1.3.1.44, such as the gene product of ter (GenbankAccession No. AAW66853.1) (Hoffmeister et al., 2005, J. Biol. Chem.,280(6), 4329-4338; Shen et al., 2011, supra) or tdter (Genbank AccessionNo. AAS11092.1) (Bond-Watts et al., Biochemistry, 2012, 51, 6827-6837).

In some embodiments, a reversible glutaconyl-CoA decarboxylase thatrelies on a Na⁺ membrane pump can be classified, for example, under EC4.1.1.70 (see Mouttaki et al., 2007, supra). The EC 4.1.1.70 enzymeactivity is associated with the following subunits in S. aciditrophicus,viz. Genbank Accession Nos. (1) ABC77900.1, (2) ABC76114.1 and (3)ABC77898.1.

In some embodiments, a glutaryl-CoA dehydrogenase from S. aciditrophicussubject to electron bifurcation via NADH can be classified under EC1.3.8.6 such as the dehydrogenases in Genbank Accession Nos. (1)ABC77899.1, (2) ABC76101.1, (3) ABC76260.1, (4) ABC76949.1 or (5)ABC78863.1.

In some embodiments, a homocitrate synthase can be classified, forexample, under EC 2.3.3.14 or EC 2.3.3.13, such as the gene product ofLYS20 and LYS21 from Saccharomyces cerevisiae or hcs from Thermusthermophiles.

In some embodiments, the combination of homocitrate dehydratase andhomoaconitate hydratase may be classified, for example, under EC 4.2.1.-(e.g., EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33), such as the geneproduct of LYS4 from Saccharomyces cerevisiae or lysT and LysU fromThermus thermophiles.

In some embodiments, an isohomocitrate dehydrogenase may be classified,for example, under EC 1.1.1.- such as EC 1.1.1.85, EC 1.1.1.87 or EC1.1.1.286, such as the gene product of LYS12 from Saccharomycescerevisiae or hicdh from Thermus thermophiles.

In some embodiments, a 5-oxopentanoate dehydrogenase may be classified,for example under EC 1.2.1.- such as the gene product of CpnE (see, forexample, Iwaki et al., 2002, Appl. Environ. Microbiol.,68(11):5671-5684).

In some embodiments, an indolepyruvate decarboxylase can be classified,for example, under EC 4.1.1.43 or EC 4.1.1.74 such as theindole-3-pyruvate decarboxylase from Salmonella typhimurium (see, forexample, Genbank Accession No. CAC48239.1 in which residue 544 can be aleucine or an alanine). A mutant variant of the indolepyruvatedecarboxylase from Salmonella typhimurium has been engineeredsuccessfully to selectively accept longer chain length substrates. TheL544A mutation of Genbank Accession No. CAC48239.1 allowed for 567 timeshigher selectivity towards the C7 2-oxoacid than towards the C52-oxoacid (see, Xiong et al., 2012, Scientific Reports, 2: 311).

In some embodiments, a 3-hydroxyadipyl-CoA dehydrogenase (also can bereferred to as 3-hydroxyacyl-CoA:NAD⁺ oxidoreductase) may be classifiedunder EC 1.1.1.157 such as the gene product of paaH (see, for example,Fuchs et al., 2011, Nature Reviews Microbiology, 9, 803-816). Homologuesof paaH in S. aciditrophicus include the 3-hydroxyacyl-CoA dehydrogenaseof Genbank Accession No. ABC77793.1.

In some embodiments, a 6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase canbe classified under EC 3.7.1.- such as the gene product of Oah or BamA(see, for example, Fuchs et al., 2011, supra).

In some embodiments, a 6-hydroxycyclohex-1-ene-1-carboxyl-CoAdehydrogenase can be classified under EC 1.1.1.- such as the geneproduct of Had or BamA (see, for example, Fuchs et al., 2011, supra).

In some embodiments, a cyclohexa-1,5-dienecarbonyl-CoA hydratase can beclassified under EC 4.2.1.100 such as the gene product of Dch or BamR(see, for example, Fuchs et al., 2011, supra).

In some embodiments, a cyclohex-1-ene-1-carboxyl-CoA dehydrogenase canbe classified under EC 1.3.8.- such as the acyl-CoA dehydrogenase ofGenbank Accession No. ABC76101.1 from S. aciditrophicus (see, forexample, Kung et al., 2013, J. Bacteriol., 195(14), 3193-3200). Thisenzyme was reassigned to class EC 1.3.8.- from EC 1.3.99.-.

In some embodiments, a cyclohex-1-ene-1-carboxyl-CoA hydratase can beclassified under EC 4.2.1.- such as the gene product of badK (see, forexample, Harwood et al., 1999, FEMS Microbiol. Reviews, 22, 439-458).Homologues of badK in S. aciditrophicus include the enoyl-CoA hydrataseof Genbank Accession No. ABC77794.1.

In some embodiments, a 2-hydroxycyclohexanecarboxyl-CoA dehydrogenasecan be classified under EC 1.1.1.- such as the gene product of badH(see, for example, Harwood et al., 1999, FEMS Microbiol. Reviews, 22,439-458). Homologs of badH in S. aciditrophicus include the3-oxoacyl-ACP reductase of Genbank Accession No. ABC76948.1.

In some embodiments, a 2-ketocyclohexanecarboxyl-CoA hydrolase can beclassified under EC 4.1.3.36 such as the gene product of badI (see, forexample, Harwood et al., 1999, supra). Homologs of badI in S.aciditrophicus such as encoded by Genbank Accession No. ABC78756.1.

Enzymes Generating the Terminal Carboxyl Groups in the Biosynthesis ofC7 Building Blocks

As depicted in FIG. 1 and FIG. 2, a terminal carboxyl group can beenzymatically formed using a thioesterase, an aldehyde dehydrogenase, a7-oxoheptanoate dehydrogenase, a 6-oxohexanoate dehydrogenase, aCoA-transferase, or a reversible CoA-ligase. In some embodiments, onlyone terminal carboxyl group is added as the central metabolite C7building block (e.g., 3-ketopimeloyl-CoA) already has a carboxyl groupat one terminus.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of a C7 building block (e.g., pimelic acid) is enzymaticallyformed by a thioesterase classified under EC 3.1.2.-, such as the geneproduct of YciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1)or Acot13 (see, for example, Cantu et al., Protein Science, 2010, 19,1281-1295; Zhuang et al., Biochemistry, 2008, 47(9), 2789-2796; orNaggert et al., J. Biol. Chem., 1991, 266(17), 11044-11050).

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by an aldehydedehydrogenase classified, for example, under EC 1.2.1.3 (see, forexample, Guerrillot & Vandecasteele, Eur. J. Biochem., 1977, 81,185-192).

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a 6-oxohexanoatedehydrogenase or a 7-oxoheptanoate dehydrogenase classified under EC1.2.1.-, such as the gene product of ChnE from Acinetobacter sp. or ThnGfrom Sphingomonas macrogolitabida (see, for example, Iwaki et al., Appl.Environ. Microbiol., 1999, 65(11), 5158-5162; or López-Sanchez et al.,Appl. Environ. Microbiol., 2010, 76(1), 110-118). For example, a6-oxohexanoate dehydrogenase can be classified under EC 1.2.1.63. Forexample, a 7-oxoheptanoate dehydrogenase can be classified under EC1.2.1.-.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a CoA-transferasesuch as a glutaconate CoA-transferase classified, for example, under EC2.8.3.12 such as from Acidaminococcus fermentans. See, for example,Buckel et al., 1981, Eur. J. Biochem., 118:315-321.

In some embodiments, a terminal carboxyl group leading to the synthesisof pimelic acid is enzymatically formed by a reversible CoA-ligaseclassified under EC 6.2.1.5.

In some embodiments, the second terminal carboxyl group leading to thesynthesis of pimelic acid is enzymatically formed by a reversibleCoA-ligase such as a succinate-CoA-ligase classified, for example, underEC 6.2.1.5 such as from Thermococcus kodakaraensis. See, for example,Shikata et al., 2007, J. Biol. Chem., 282(37):26963-26970.

Enzymes Generating the Terminal Amine Groups in the Biosynthesis of C7Building Blocks

As depicted in FIG. 3 and FIG. 4, terminal amine groups can beenzymatically formed using a ω-transaminase or a deacetylase.

In some embodiments, the first terminal amine group leading to thesynthesis of 7-aminoheptanoic acid is enzymatically formed by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as that obtained fromChromobacterium violaceum (Genbank Accession No. AAQ59697.1, SEQ ID NO:8), Pseudomonas aeruginosa (Genbank Accession No. AAG08191.1, SEQ ID NO:9), Pseudomonas syringae (Genbank Accession No. AAY39893.1, SEQ ID NO:10), Rhodobacter sphaeroides (Genbank Accession No. ABA81135.1, SEQ IDNO: 11), Vibrio Fluvialis (Genbank Accession No. AAA57874.1, SEQ ID NO:13), Streptomyces griseus, or Clostridium viride. An additionalω-transaminase that can be used in the methods and hosts describedherein is from Escherichia coli (Genbank Accession No. AAA57874.1, SEQID NO: 12). Some of the ω-transaminases classified, for example, underEC 2.6.1.29 or EC 2.6.1.82 are diamine ω-transaminases (e.g., SEQ IDNO:12).

The reversible ω-transaminase from Chromobacterium violaceum (GenbankAccession No. AAQ59697.1, SEQ ID NO: 8) has demonstrated analogousactivity accepting 6-aminohexanoic acid as amino donor, thus forming thefirst terminal amine group in adipate semialdehyde (Kaulmann et al.,Enzyme and Microbial Technology, 2007, 41, 628-637).

The reversible 4-aminobubyrate:2-oxoglutarate transaminase fromStreptomyces griseus has demonstrated analogous activity for theconversion of 6-aminohexanoate to adipate semialdehyde (Yonaha et al.,Eur. J. Biochem., 1985, 146, 101-106).

The reversible 5-aminovalerate transaminase from Clostridium viride hasdemonstrated analogous activity for the conversion of 6-aminohexanoateto adipate semialdehyde (Barker et al., J. Biol. Chem., 1987, 262(19),8994-9003).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by a diaminetransaminase. For example, the second terminal amino group can beenzymatically formed by a diamine transaminase classified, for example,under EC 2.6.1.29 or classified, for example, under EC 2.6.1.82, such asthe gene product of YgjG from E. coli (Genbank Accession No. AAA57874.1,SEQ ID NO: 12).

The gene product of ygjG accepts a broad range of diamine carbon chainlength substrates, such as putrescine, cadaverine and spermidine(Samsonova et al., BMC Microbiology, 2003, 3:2).

The diamine transaminase from E. coli strain B has demonstrated activityfor 1,7 diaminoheptane (Kim, The Journal of Chemistry, 1964, 239(3),783-786).

In some embodiments, the second terminal amine group leading to thesynthesis of heptamethylenediamine is enzymatically formed by adeacetylase classified, for example, under EC 3.5.1.62 such as anacetylputrescine deacetylase. The acetylputrescine deacetylase fromMicrococcus luteus K-11 accepts a broad range of carbon chain lengthsubstrates, such as acetylputrescine, acetylcadaverine andN⁸-acetylspermidine (see, for example, Suzuki et al., 1986, BBA—GeneralSubjects, 882(1):140-142).

Enzymes Generating the Terminal Hydroxyl Groups in the Biosynthesis ofC7 Building Blocks

As depicted in FIG. 5 and FIG. 6, the terminal hydroxyl group can beenzymatically formed using an alcohol dehydrogenase.

In some embodiments, the terminal hydroxyl group leading to thesynthesis of 1,7 heptanediol is enzymatically formed by an alcoholdehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC1.1.1.1, 1.1.1.2, 1.1.1.21, or 1.1.1.184).

Biochemical Pathways

Pathway Using Acetyl-CoA or 2-Oxo-Glutarate as Central Metabolite in theBiosynthesis of C7 Backbone

In some embodiments, glutaryl-CoA is synthesized from the centralmetabolite, acetyl-CoA, by conversion of acetyl-CoA to acetoacetyl-CoAby a β-ketothiolase classified, for example, under EC 2.3.1.9 such asthe gene product of atoB or phaA or by an acetyl-CoA carboxylaseclassified under, for example, EC 6.4.1.2 and an acetoacetyl-CoAsynthase classified, for example, under EC 2.3.1.194; followed byconversion to 3-hydroxybutanoyl-CoA by a 3-hydroxyacyl-CoA dehydrogenaseclassified, for example, under EC 1.1.1.- such as EC 1.1.1.157 such asthe gene product of hbd; followed by conversion to crotonyl-CoA by anenoyl-CoA reductase classified, for example, under EC 4.2.1.- (e.g., EC4.2.1.17) such as the gene product of crt, followed by conversion toeither a) glutaconyl-CoA by a glutaconyl-CoA decarboxylase classified,for example, under EC 4.1.1.70; followed by conversion to glutaryl-CoAby either (i) an enoyl-[acp] reductase classified, for example, under EC1.3.1.9 or (ii) a trans-2-enoyl-CoA reductase classified, for example,under EC 1.3.1.44 such as the gene product of ter or tdter or (b)glutaryl-CoA by a glutaryl-CoA dehydrogenase subject to electronbifurcation from Synthrophus aciditrophicus such as the dehydrogenasesof Genbank Accession Nos. (1) ABC77899.1, (2) ABC76101.1, (3)ABC76260.1, (4) ABC76949.1 or (5) ABC78863.1. See, FIG. 1.

In some embodiments, glutaryl-CoA can be synthesized from the centralmetabolite, 2-oxoglutarate, by conversion of 2-oxoglutarate to(Homo)₁citrate by a homocitrate synthase classified, for example, underEC 2.3.3.14 or EC 2.3.3.13 such as the gene product of LYS20 and LYS21from Saccharomyces cerevisiae or hcs from Thermus thermophiles; followedby conversion to iso(homo)₁citrate by a homocitrate dehydratase and ahomoaconitate hydratase classified, for example, under EC 4.2.1.114, EC4.2.1.36 or EC 4.2.1.33 such as the gene product of LYS4 fromSaccharomyces cerevisiae or lysT and LysU from Thermus thermophiles;followed by conversion to 2-oxoadipate by an iso(homo)_(n)citratedehydrogenase classified, for example, under EC 1.1.1.85, EC 1.1.1.87 orEC 1.1.1.286 such as the gene product of LYS12 from Saccharomycescerevisiae or hicdh from Thermus thermophiles; followed by conversion toglutarate semialdehyde by an indolepyruvate decarboxylase classifiedunder EC 4.1.1.43 or EC 4.1.1.74 (e.g., GenBank Accession No.CAC48239.1); followed by conversion to glutarate by a glutaratesemialdehyde dehydrogenase classified, for example, under EC 1.2.1.20,EC 1.2.1.16 or EC 1.2.1.79 such as the gene product of CpnE; followed byconversion to glutaryl-CoA by a glutarate:CoA ligase classified, forexample, under EC 6.2.1.6. See, e.g., FIG. 1.

In some embodiments, pimeloyl-CoA can be synthesized from glutaryl-CoAproduced as described above by conversion of glutaryl-CoA to3-ketopimeloyl-CoA by a β-ketothiolase classified under EC 2.3.1.-,e.g., EC 2.3.1.174 or EC 2.3.1.16 such as the gene product of paaJ orhomologs of paaJ (e.g., Genbank Accession No. ABC78517.1 or GenbankAccession No. ABC78881.1) or by a β-ketoacyl-[acp] synthase classified,for example, under EC 2.3.1.41, EC 2.3.1.179, EC 2.3.1.180; followed byconversion to 3-hydroxypimeloyl-CoA by a 3-hydroxyadipyl-CoAdehydrogenase classified, for example, under EC 1.1.1.157 such as thegene product of paaH or homologs of paaH (e.g., Genbank Accession No.ABC77793.1); followed by conversion to 2,3-dehydropimeloyl-CoA by anenoyl-CoA hydratase such as the gene product of paaF or homologs of paaF(e.g., Genbank Accession No. ABC77794.1 or Genbank Accession No.ABC78950.1); followed by conversion to pimeloyl-CoA by an enoyl-[acp]reductase from S. aciditrophicus classified, for example, under EC1.3.1.9 or a trans-2-enoyl-CoA reductase classified, for example, underEC 1.3.1.44 such as the gene product of ter or tdter. See, e.g., FIG. 1.

In some embodiments, pimeloyl-CoA can be synthesized from glutaryl-CoAproduced as described above by conversion of glutaryl-CoA to3-ketopimeloyl-CoA by a β-ketothiolase classified under EC 2.3.1.-,e.g., EC 2.3.1.174 or EC 2.3.1.16 such as the gene product of paaJ orhomologs of paaJ (e.g., Genbank Accession No. ABC78517.1 or GenbankAccession No. ABC78881.1) or by a β-ketoacyl-[acp] synthase classified,for example, under EC 2.3.1.41, EC 2.3.1.179, EC 2.3.1.180; followed byconversion to 6-ketocyclohex-1-ene-1-carboxyl-CoA by a6-oxo-cyclohex-1-ene-carbonyl-CoA hydrolase classified, for example,under EC 3.7.1.- such as the gene product of Oah or BamA; followed byconversion to 6-hydroxycyclohex-1-ene-1-carboxyl-CoA by a6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified, forexample, under EC 1.1.1.- such as the gene product of Had or BamQ;followed by conversion to cyclohexa-1,5-diene-1-carbonyl-CoA by acyclohexa-1,5-dienecarbonyl-CoA hydratase classified under, for example,EC 4.2.1.100 such as the gene product of Dch or BamR; followed byconversion to cyclohex-1-ene-1-carboxyl-CoA by acyclohex-1-ene-1-Carboxyl-CoA dehydrogenase classified under EC 1.3.8.-such as Genbank Accession No. ABC76100.1; followed by conversion to2-hydroxycyclohexane-1-carboxyl-CoA by a cyclohex-1-ene-1-carboxyl-CoAhydratase classified under, for example, EC 4.2.1.- such as the geneproduct of badK or homologs of badK (e.g., Genbank Accession No.ABC77794.1); followed by conversion to2-ketocyclohex-1-ene-1-carboxyl-CoA by a2-hydroxycyclohexanecarboxyl-CoA dehydrogenase classified, for example,under EC 1.1.1.- such as the gene product of badH or homologs of badH(e.g., Genbank Accession No. ABC76948.1); followed by conversion topimeloyl-CoA by a 2-ketocyclohexanecarboxyl-CoA hydrolase classified,for example, under, EC 4.1.3.36 such as the gene product of badI orhomologs of badI (e.g., Genbank Accession No. ABC78756.1).

Pathways Using Pimeloyl-CoA as Central Precursor to Pimelic Acid

In some embodiments, pimelic acid is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by athioesterase classified under EC 3.1.2.- such as the gene products ofYciA, tesB (Genbank Accession No. AAA24665.1, SEQ ID NO: 1) or Acot13.See FIG. 2.

In some embodiments, pimelic acid is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by areversible CoA ligase such as a succinate CoA-ligase classified, forexample, under EC 6.2.1.5. See FIG. 2.

In some embodiments, pimelic acid is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelate by aCoA-transferase classified, for example, under EC 2.8.3.12 such as aglutaconate CoA-transferase. See FIG. 2.

In some embodiments, pimeloyl-CoA can be converted to pimelatesemialdehyde by an acetylating aldehyde dehydrogenase classified, forexample, under EC 1.2.1.10 such as the gene product of PduB or PduP;followed by conversion to pimelic acid by a dehydrogenase classified,for example, under EC 1.2.1.- such as a 7-oxoheptanoate dehydrogenase(e.g., the gene product of ThnG), a 6-oxohexanoate dehydrogenase (e.g.,the gene product of ChnE), or an aldehyde dehydrogenase classified underEC 1.2.1.3. See FIG. 2.

Pathways Using Pimeloyl-CoA as Central Precursor to 7-Aminoheptanoate

In some embodiments, 7-aminoheptanoate is synthesized from the centralprecursor, pimelate or pimeloyl-CoA. For example, pimelate, formed asdescribed above, can be converted to pimelate semialdehyde by acarboxylate reductase classified, for example, under EC 1.2.99.6 such asthe gene product of car in combination with a phosphopantetheinetransferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilisor npt gene from Nocardia) or the gene products of GriC and GriD fromStreptomyces griseus (see Suzuki et al., J. Antibiot., 2007, 60(6),380-387); followed by conversion of pimelate semialdehyde to7-aminoheptanoate by a ω-transaminase (classified, for example, under EC2.6.1.- EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.48, EC 2.6.1.82, see SEQ IDNOs:8-13). See FIG. 3. The carboxylate reductase can be obtained, forexample, from Mycobacterium marinum (Genbank Accession No. ACC40567.1,SEQ ID NO: 2), Mycobacterium smegmatis (Genbank Accession No.ABK71854.1, SEQ ID NO: 3), Segniliparus rugosus (Genbank Accession No.EFV11917.1, SEQ ID NO: 4), Mycobacterium smegmatis (Genbank AccessionNo. ABK75684.1, SEQ ID NO: 5), Mycobacterium massiliense (GenbankAccession No. EIV11143.1, SEQ ID NO: 6), or Segniliparus rotundus(Genbank Accession No. ADG98140.1, SEQ ID NO: 7). See FIGS. 7A-7F.

The carboxylate reductase encoded by the gene product of car andenhancer npt or sfp has broad substrate specificity, including terminaldifunctional C4 and C5 carboxylic acids (Venkitasubramanian et al.,Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, pimeloyl-CoA formed as described above is convertedto pimelate semialdehyde by an acetylating aldehyde dehydrogenaseclassified, for example, under EC 1.2.1.10 such as the gene product ofPduB or PduP; followed by conversion of pimelate semialdehyde to7-aminoheptanoate by a ω-transaminase classified, for example, under EC2.6.1.- such as EC 2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48, see). See FIG.3.

Pathway Using 7-Aminoheptanoate as Central Precursor toHeptamethylenediamine

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto 7-aminoheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus (Suzukiet al., 2007, supra); followed by conversion of 7-aminoheptanal toheptamethylenediamine by a ω-transaminase (e.g., EC 2.6.1.18, EC2.6.1.19, EC 2.6.1.48, EC 2.6.1.82 such as SEQ ID NOs:8-13). See FIG. 4.

The carboxylate reductase encoded by the gene product of car andenhancer npt has broad substrate specificity, including terminaldifunctional C4 and C5 carboxylic acids (Venkitasubramanian et al.,Enzyme and Microbial Technology, 2008, 42, 130-137).

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-hydroxyheptanoate (which can be produced asdescribed in FIG. 5), by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD (Suzuki et al., 2007, supra);followed by conversion of 7-aminoheptanal to 7-aminoheptanol by aω-transaminase classified, for example, under EC 2.6.1.18, EC 2.6.1.19,EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, seeabove; followed by conversion to 7-aminoheptanal by an alcoholdehydrogenase classified, for example, under EC 1.1.1.- (e.g., EC1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184) such as the geneproduct of YMR318C or YqhD (Liu et al., Microbiology, 2009, 155,2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; Jarboe,2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or the proteinhaving GenBank Accession No. CAA81612.1; followed by conversion toheptamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas SEQ ID NOs:8-13, see above. See FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, 7-aminoheptanoate, by conversion of 7-aminoheptanoateto N7-acetyl-7-aminoheptanoate by an N-acetyltransferase such as alysine N-acetyltransferase classified, for example, under EC 2.3.1.32;followed by conversion to N7-acetyl-7-aminoheptanal by a carboxylatereductase classified, for example, under EC 1.2.99.6 such as the geneproduct of car (see above) in combination with a phosphopantetheinetransferase enhancer (e.g., encoded by a sfp gene from Bacillus subtilisor npt gene from Nocardia) or the gene product of GriC & GriD; followedby conversion to N7-acetyl-1,7-diaminoheptane by a ω-transaminaseclassified, for example, under EC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC2.6.1.48, or EC 2.6.1.82 such as SEQ ID NOs:8-13, see above; followed byconversion to heptamethylenediamine by an acetylputrescine deacetylaseclassified, for example, under EC 3.5.1.62. See, FIG. 4.

In some embodiments, heptamethylenediamine is synthesized from thecentral precursor, pimelate semialdehyde, by conversion of pimelatesemialdehyde to heptanedial by a carboxylate reductase classified, forexample, under EC 1.2.99.6 such as the gene product of car (see above)in combination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene product of GriC & GriD; followed by conversion to7-aminoheptanal by a ω-transaminase classified, for example, under EC2.6.1.18, EC 2.6.1.19, or EC 2.6.1.48; followed by conversion toheptamethylenediamine by a ω-transaminase classified, for example, underEC 2.6.1.18, EC 2.6.1.19, EC 2.6.1.29, EC 2.6.1.48, or EC 2.6.1.82 suchas SEQ ID NOs:8-13. See FIG. 4.

Pathways Using Pimelate or Pimelate Semialdehyde as Central Precursorsto 7-Hydroxyheptanoate or 1,7-Heptanediol

In some embodiments, 7-hydroxyheptanoate is synthesized from the centralprecursor, pimelate, by conversion of pimelate to pimelate semialdehydeby a carboxylate reductase classified, for example, under EC 1.2.99.6such as the gene product of car (see above) in combination with aphosphopantetheine transferase enhancer (e.g., encoded by a sfp genefrom Bacillus subtilis or npt gene from Nocardia) or the gene product ofGriC & GriD; followed by conversion to 7-hydroxyheptanoate by a6-hydroxyhexanoate dehydrogenase classified, for example, under EC1.1.1.258 such as the gene product of ChnD, a 5-hydroxypentanoatedehydrogenase classified, for example, under EC 1.1.1.- such as the geneproduct of CpnD (see, for example, Iwaki et al., 2002, Appl. Environ.Microbiol., 68(11):5671-5684) or a 4-hydroxybutyrate dehydrogenase suchas gabD (see, for example, Lütke-Eversloh & Steinbüchel, 1999, FEMSMicrobiology Letters, 181(1):63-71). See FIG. 5.

In some embodiments, 7-hydroxyheptanoate is synthesized from the centralprecursor, pimeloyl-CoA, by conversion of pimeloyl-CoA to pimelatesemialdehyde by an acetylating aldehyde dehydrogenase classified, forexample, under EC 1.2.1.10 such as the gene product of PduB or PduP;followed by conversion to 7-hydroxyheptanoate by a 6-hydroxyhexanoatedehydrogenase classified, for example, under EC 1.1.1.258 such as thegene product of ChnD, a 5-hydroxypentanoate dehydrogenase classified,for example, under EC 1.1.1.- such as the gene product of CpnD (see, forexample, Iwaki et al., 2002, Appl. Environ. Microbiol.,68(11):5671-5684) or a 4-hydroxybutyrate dehydrogenase such as gabD(see, for example, Lüke-Eversloh & Steinbüchel, 1999, FEMS MicrobiologyLetters, 181(1):63-71). See FIG. 5.

In some embodiments, pimeloyl-CoA formed as described above is convertedto pimelate semialdehyde by an acetylating aldehyde dehydrogenase(classified, for example, under EC 1.2.1.10) such as the gene product ofPduB; followed by conversion of pimelate semialdehyde to7-hydroxyheptanoate by an alcohol dehydrogenase classified for example,under EC 1.1.1.- (e.g., EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC1.1.1.184). See FIG. 5.

In some embodiments, 1,7 heptanediol is synthesized from the centralprecursor, 7-hydroxyheptanoate, by conversion of 7-hydroxyheptanoate to7-hydroxyheptanal by a carboxylate reductase classified, for example,under EC 1.2.99.6 such as the gene product of car (see above) incombination with a phosphopantetheine transferase enhancer (e.g.,encoded by a sfp gene from Bacillus subtilis or npt gene from Nocardia)or the gene products of GriC and GriD from Streptomyces griseus (Suzukiet al., 2007, supra); followed by conversion of 7-hydroxyheptanal to 1,7heptanediol by an alcohol dehydrogenase classified, for example, underEC 1.1.1.- such as EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.21, or EC 1.1.1.184)such as the gene product of YMR318C or YqhD (from E. coli, GenBankAccession No. AAA69178.1) (see, e.g., Liu et al., Microbiology, 2009,155, 2078-2085; Larroy et al., 2002, Biochem J., 361(Pt 1), 163-172; orJarboe, 2011, Appl. Microbiol. Biotechnol., 89(2), 249-257) or theprotein having GenBank Accession No. CAA81612.1 (from Geobacillusstearothermophilus). See FIG. 6.

Cultivation Strategy

In some embodiments, the cultivation strategy entails achieving ananaerobic cultivation, micro-aerobic, or mixed oxygen/denitrificationcondition. Enzymes characterized in vitro as being oxygen sensitiverequire a micro-aerobic cultivation strategy maintaining a very lowdissolved oxygen concentration (See, for example, Chayabatra & Lu-Kwang,Appl. Environ. Microbiol., 2000, 66(2), 493 0 498; Wilson and Bouwer,1997, Journal of Industrial Microbiology and Biotechnology, 18(2-3),116-130).

In some embodiments, the cultivation strategy entails nutrientlimitation such as nitrogen, phosphate or oxygen limitation.

In some embodiments, a final electron acceptor other than oxygen such asnitrates can be utilized.

In some embodiments, a cell retention strategy using, for example,ceramic hollow fiber membranes can be employed to achieve and maintain ahigh cell density during either fed-batch or continuous fermentation.

In some embodiments, the principal carbon source fed to the fermentationin the synthesis of one or more C7 building blocks can derive frombiological or non-biological feedstocks.

In some embodiments, the biological feedstock can be, can include, orcan derive from, monosaccharides, disaccharides, lignocellulose,hemicellulose, cellulose, lignin, levulinic acid and formic acid,triglycerides, glycerol, fatty acids, agricultural waste, condenseddistillers' solubles, or municipal waste.

The efficient catabolism of crude glycerol stemming from the productionof biodiesel has been demonstrated in several microorganisms such asEscherichia coli, Cupriavidus necator, Pseudomonas oleavorans,Pseudomonas putida and Yarrowia lipolytica (Lee et al., Appl. Biochem.Biotechnol., 2012, 166, 1801-1813; Yang et al., Biotechnology forBiofuels, 2012, 5:13; Meijnen et al., Appl. Microbiol. Biotechnol.,2011, 90, 885-893).

The efficient catabolism of lignocellulosic-derived levulinic acid hasbeen demonstrated in several organisms such as Cupriavidus necator andPseudomonas putida in the synthesis of 3-hydroxyvalerate via theprecursor propanoyl-CoA (Jaremko and Yu, Journal of Biotechnology, 2011,155, 2011, 293-298; Martin and Prather, Journal of Biotechnology, 2009,139, 61-67).

The efficient catabolism of lignin-derived aromatic compounds such asbenzoate analogues has been demonstrated in several microorganisms suchas Pseudomonas putida, Cupriavidus necator (Bugg et al., Current Opinionin Biotechnology, 2011, 22, 394-400; Pérez-Pantoja et al., FEMSMicrobiol. Rev., 2008, 32, 736-794).

The efficient utilization of agricultural waste, such as olive millwaste water has been demonstrated in several microorganisms, includingYarrowia lipolytica (Papanikolaou et al., Bioresour. Technol., 2008,99(7), 2419-2428).

The efficient utilization of fermentable sugars such as monosaccharidesand disaccharides derived from cellulosic, hemicellulosic, cane and beetmolasses, cassava, corn and other agricultural sources has beendemonstrated for several microorganism such as Escherichia coli,Corynebacterium glutamicum and Lactobacillus delbrueckii and Lactococcuslactis (see, e.g., Hermann et al, Journal of Biotechnology, 2003, 104,155-172; Wee et al., Food Technol. Biotechnol., 2006, 44(2), 163-172;Ohashi et al., Journal of Bioscience and Bioengineering, 1999, 87(5),647-654).

The efficient utilization of furfural, derived from a variety ofagricultural lignocellulosic sources, has been demonstrated forCupriavidus necator (Li et al., Biodegradation, 2011, 22, 1215-1225).

In some embodiments, the non-biological feedstock can be or can derivefrom natural gas, syngas, CO₂/H₂, methanol, ethanol, benzoic acid,non-volatile residue (NVR), a caustic wash waste stream from cyclohexaneoxidation processes, or terephthalic acid/isophthalic acid mixture wastestreams.

The efficient catabolism of methanol has been demonstrated for themethylotrophic yeast Pichia pastoris.

The efficient catabolism of ethanol has been demonstrated forClostridium kluyveri (Seedorf et al., Proc. Natl. Acad. Sci. USA, 2008,105(6) 2128-2133).

The efficient catabolism of CO₂ and H₂, which may be derived fromnatural gas and other chemical and petrochemical sources, has beendemonstrated for Cupriavidus necator (Prybylski et al., Energy,Sustainability and Society, 2012, 2:11).

The efficient catabolism of syngas has been demonstrated for numerousmicroorganisms, such as Clostridium ljungdahlii and Clostridiumautoethanogenum (Köpke et al., Applied and Environmental Microbiology,2011, 77(15), 5467-5475).

The efficient catabolism of the non-volatile residue waste stream fromcyclohexane processes has been demonstrated for numerous microorganisms,such as Delftia acidovorans and Cupriavidus necator (Ramsay et al.,Applied and Environmental Microbiology, 1986, 52(1), 152-156).

In some embodiments, the host microorganism is a prokaryote. Forexample, the prokaryote can be a bacterium from the genus Escherichiasuch as Escherichia coli; from the genus Clostridia such as Clostridiumljungdahlii, Clostridium autoethanogenum or Clostridium kluyveri; fromthe genus Corynebacteria such as Corynebacterium glutamicum; from thegenus Cupriavidus such as Cupriavidus necator or Cupriavidusmetallidurans; from the genus Pseudomonas such as Pseudomonasfluorescens, Pseudomonas putida or Pseudomonas oleavorans; from thegenus Delftia such as Delftia acidovorans; from the genus Bacillus suchas Bacillus subtillis; from the genus Lactobacillus such asLactobacillus delbrueckii; or from the genus Lactococcus such asLactococcus lactis. Such prokaryotes also can be a source of genes toconstruct recombinant host cells described herein that are capable ofproducing one or more C7 building blocks.

In some embodiments, the host microorganism is a eukaryote. For example,the eukaryote can be a filamentous fungus, e.g., one from the genusAspergillus such as Aspergillus niger. Alternatively, the eukaryote canbe a yeast, e.g., one from the genus Saccharomyces such as Saccharomycescerevisiae; from the genus Pichia such as Pichia pastoris; or from thegenus Yarrowia such as Yarrowia lipolytica; from the genus Issatchenkiasuch as Issathenkia orientalis; from the genus Debaryomyces such asDebaryomyces hansenii; from the genus Arxula such as Arxulaadenoinivorans; or from the genus Kluyveromyces such as Kluyveromyceslactis. Such eukaryotes also can be a source of genes to constructrecombinant host cells described herein that are capable of producingone or more C7 building blocks.

Metabolic Engineering

The present document provides methods involving less than all the stepsdescribed for all the above pathways. Such methods can involve, forexample, one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve or more of such steps. Where less than all the steps areincluded in such a method, the first, and in some embodiments the only,step can be any one of the steps listed.

Furthermore, recombinant hosts described herein can include anycombination of the above enzymes such that one or more of the steps,e.g., one, two, three, four, five, six, seven, eight, nine, ten, or moreof such steps, can be performed within a recombinant host. This documentprovides host cells of any of the genera and species listed andgenetically engineered to express one or more (e.g., two, three, four,five, six, seven, eight, nine, 10, 11, 12 or more) recombinant forms ofany of the enzymes recited in the document. Thus, for example, the hostcells can contain exogenous nucleic acids encoding enzymes catalyzingone or more of the steps of any of the pathways described herein.

In addition, this document recognizes that where enzymes have beendescribed as accepting CoA-activated substrates, analogous enzymeactivities associated with [acp]-bound substrates exist that are notnecessarily in the same enzyme class.

Also, this document recognizes that where enzymes have been describedaccepting (R)-enantiomers of substrate, analogous enzyme activitiesassociated with (S)-enantiomer substrates exist that are not necessarilyin the same enzyme class.

This document also recognizes that where an enzyme is shown to accept aparticular co-factor, such as NADPH, or a co-substrate, such asacetyl-CoA, many enzymes are promiscuous in terms of accepting a numberof different co-factors or co-substrates in catalyzing a particularenzyme activity. Also, this document recognizes that where enzymes havehigh specificity for e.g., a particular co-factor such as NADH, anenzyme with similar or identical activity that has high specificity forthe co-factor NADPH may be in a different enzyme class.

In some embodiments, the enzymes in the pathways outlined herein are theresult of enzyme engineering via non-direct or rational enzyme designapproaches with aims of improving activity, improving specificity,reducing feedback inhibition, reducing repression, improving enzymesolubility, changing stereo-specificity, or changing co-factorspecificity.

In some embodiments, the enzymes in the pathways outlined herein can begene dosed (i.e., overexpressed by having a plurality of copies of thegene in the host organism), into the resulting genetically modifiedorganism via episomal or chromosomal integration approaches.

In some embodiments, genome-scale system biology techniques such as FluxBalance Analysis can be utilized to devise genome scale attenuation orknockout strategies for directing carbon flux to a C7 building block.

Attenuation strategies include, but are not limited to; the use oftransposons, homologous recombination (double cross-over approach),mutagenesis, enzyme inhibitors and RNA interference (RNAi).

In some embodiments, fluxomic, metabolomic and transcriptomal data canbe utilized to inform or support genome-scale system biology techniques,thereby devising genome scale attenuation or knockout strategies indirecting carbon flux to a C7 building block.

In some embodiments, the host microorganism's tolerance to highconcentrations of a C7 building block can be improved through continuouscultivation in a selective environment.

In some embodiments, the host microorganism's endogenous biochemicalnetwork can be attenuated or augmented to ensure the intracellularavailability of acetyl-CoA (2) create an NADH imbalance that may only bebalanced via the formation of a C7 building block, (3) preventdegradation of central metabolites or central precursors leading to andincluding C7 building blocks and (4) ensure efficient efflux from thecell.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenous gene encodinga phosphotransacetylase, which generates acetate, such as pta isattenuated (Shen et al., Appl. Environ. Microbiol., 2011, 77(9),2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, an endogenous gene encodingan acetate kinase in an acetate synthesis pathway, such as ack, isattenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of pyruvate to lactatesuch as ldhA is attenuated (Shen et al., Appl. Environ. Microbiol.,2011, 77(9), 2905-2915).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of phophoenolpyruvateto succinate such as frdBC is attenuated (see, e.g., Shen et al., 2011,supra).

In some embodiments requiring the intracellular availability ofacetyl-CoA and NADH for C7 building block synthesis, an endogenous geneencoding an enzyme that catalyzes the degradation of acetyl-CoA toethanol such as the alcohol dehydrogenase encoded by adhE is attenuated(Shen et al., 2011, supra).

In some embodiments, an endogenous gene encoding an enzyme thatcatalyzes the degradation of pyruvate to ethanol such as pyruvatedecarboxylase is attenuated.

In some embodiments requiring the intracellular availability ofacetyl-CoA for C7 building block synthesis, a gene encoding anacetyl-CoA synthetase such as the gene acs is overexpressed in themicroorganism (Satoh et al., Journal of Bioscience and Bioengineering,2003, 95(4), 335-341).

In some embodiments, where a pathway requires excess NADH for C7building block synthesis, carbon flux is directed into the pentosephosphate cycle by attenuating an endogenous gene encoding aglucose-6-phosphate isomerase (EC 5.3.1.9).

In some embodiments, where a pathway requires excess NADH co-factor forC7 building block synthesis, a gene encoding a formate dehydrogenase isoverexpressed in the host organism (Shen et al., 2011, supra).

In some embodiments, endogenous enzymes facilitating the conversion ofNADH to NADPH are attenuated, such as the NADPH generation cycle thatmay be generated via inter-conversion of glutamate dehydrogenases in EC1.4.1.2 (NADH-specific) and EC 1.4.1.4 (NADPH-specific). Similarly,transhydrogenases such as classified, for example, under EC 1.6.1.1, EC1.6.1.2 or EC 1.6.1.3, may be attenuated.

In some embodiments, an endogenous gene encoding a glutamatedehydrogenase (EC 1.4.1.3) that utilizes both NADH and NADPH asco-factors is attenuated.

In some embodiments, membrane-bound enoyl-CoA reductases are solubilizedvia truncation of the amino acid residues that anchor the protein in themembrane (Hoffmeister et al., J. Biol. Chem., 2005, 280(6) 4329-4338).

In some embodiments, membrane-bound enoyl-CoA reductases are solubilizedvia expression as a fusion protein (Gloerich et al., FEBS Letters, 2006,580, 2092-2096).

In some embodiments, an enoyl-[acp] reductase from fatty acid synthesisor a trans-2-enoyl-CoA reductase is subjected to enzyme engineering withthe aim of modifying the tunnel-like hydrophobic cavity to accept CoAactivated dicarboxylic acids as substrate (Roujeinikova et al., J. Mol.Biol., 2007, 365, 135-145).

In some embodiments using hosts that naturally accumulatepolyhydroxyalkanoates, an endogenous gene encoding a polymer synthaseenzyme can be attenuated in the host strain.

In some embodiments, a L-alanine dehydrogenase can be overexpressed inthe host to regenerate L-alanine from pyruvate as amino donor forω-transaminase reactions.

In some embodiments, a NADH-specific L-glutamate dehydrogenase can beoverexpressed in the host to regenerate L-glutamate from 2-oxoglutarateas amino donor for ω-transaminase reactions.

In some embodiments, enzymes such as pimeloyl-CoA dehydrogenaseclassified under, for example, EC 1.3.1.62 and/or an acyl-CoAdehydrogenase classified under, for example, EC 1.3.8.7 or EC 1.3.8.1that degrade central metabolites and central precursors leading to andincluding C7 building blocks can be attenuated.

In some embodiments, endogenous enzymes activating C7 building blocksvia Coenzyme A esterification such as CoA-ligases such as pimeloyl-CoAsynthetase classified under, for example, EC 6.2.1.14 can be attenuated.

In some embodiments, the efflux of a C7 building block across the cellmembrane to the extracellular media can be enhanced or amplified bygenetically engineering structural modifications to the cell membrane orincreasing any associated transporter activity for a C7 building block.

The efflux of heptamethylenediamine can be enhanced or amplified byoverexpressing broad substrate range multidrug transporters such as Bltfrom Bacillus subtilis (Woolridge et al., 1997, J. Biol. Chem.,272(14):8864-8866); AcrB and AcrD from Escherichia coli (Elkins &Nikaido, 2002, J. Bacteriol., 184(23), 6490-6499) or NorA fromStaphylococcus aereus (Ng et al., 1994, Antimicrob Agents Chemother,38(6), 1345-1355) or Bmr from Bacillus subtilis (Neyfakh, 1992,Antimicrob Agents Chemother, 36(2), 484-485).

The efflux of 7-aminoheptanoate and heptamethylenediamine can beenhanced or amplified by overexpressing the solute transporters such asthe lysE transporter from Corynebacterium glutamicum (Bellmann et al.,2001, Microbiology, 147, 1765-1774).

The efflux of pimelic acid can be enhanced or amplified byoverexpressing a dicarboxylate transporter such as the SucE transporterfrom Corynebacterium glutamicum (Huhn et al., Appl. Microbiol. &Biotech., 89(2), 327-335).

Producing C7 Building Blocks Using a Recombinant Host

Typically, one or more C7 building blocks can be produced by providing ahost microorganism and culturing the provided microorganism with aculture medium containing a suitable carbon source as described above.In general, the culture media and/or culture conditions can be such thatthe microorganisms grow to an adequate density and produce a C7 buildingblock efficiently. For large-scale production processes, any method canbe used such as those described elsewhere (Manual of IndustrialMicrobiology and Biotechnology, 2^(nd) Edition, Editors: A. L. Demainand J. E. Davies, ASM Press; and Principles of Fermentation Technology,P. F. Stanbury and A. Whitaker, Pergamon). Briefly, a large tank (e.g.,a 100 gallon, 200 gallon, 500 gallon, or more tank) containing anappropriate culture medium is inoculated with a particularmicroorganism. After inoculation, the microorganism is incubated toallow biomass to be produced. Once a desired biomass is reached, thebroth containing the microorganisms can be transferred to a second tank.This second tank can be any size. For example, the second tank can belarger, smaller, or the same size as the first tank. Typically, thesecond tank is larger than the first such that additional culture mediumcan be added to the broth from the first tank. In addition, the culturemedium within this second tank can be the same as, or different from,that used in the first tank.

Once transferred, the microorganisms can be incubated to allow for theproduction of a C7 building block. Once produced, any method can be usedto isolate C7 building blocks. For example, C7 building blocks can berecovered selectively from the fermentation broth via adsorptionprocesses. In the case of pimelic acid and 7-aminoheptanoic acid, theresulting eluate can be further concentrated via evaporation,crystallized via evaporative and/or cooling crystallization, and thecrystals recovered via centrifugation. In the case ofheptamethylenediamine and 1,7-heptanediol, distillation may be employedto achieve the desired product purity.

The invention is further described in the following example, which doesnot limit the scope of the invention described in the claims.

EXAMPLES EXAMPLE 1 Enzyme Activity of Thioesterases Using Pimeloyl-CoAas a Substrate and Forming Pimelic Acid

A sequence encoding an N-terminal His tag was added to the tesB genefrom Escherichia coli that encodes a thioesterase (SEQ ID NO 1, see FIG.7A), such that an N-terminal HIS tagged thioesterase could be produced.The modified tesB gene was cloned into a pET15b expression vector undercontrol of the T7 promoter. The expression vector was transformed into aBL21[DE3] E. coli host. The resulting recombinant E. coli strain wascultivated at 37° C. in a 500 mL shake flask culture containing 50 mLLuria Broth (LB) media and antibiotic selection pressure, with shakingat 230 rpm. The culture was induced overnight at 17° C. using 0.5 mMIPTG.

The pellet from the induced shake flask culture was harvested viacentrifugation. The pellet was resuspended and lysed in Y-Per™ solution(ThermoScientific, Rockford, Ill.). The cell debris was separated fromthe supernatant via centrifugation. The thioesterase was purified fromthe supernatant using Ni-affinity chromatography and the eluate wasbuffer exchanged and concentrated via ultrafiltration.

The enzyme activity assay was performed in triplicate in a buffercomposed of 50 mM phosphate buffer (pH=7.4), 0.1 mM Ellman's reagent,and 667 μM of pimeloyl-CoA (as substrate). The enzyme activity assayreaction was initiated by adding 0.8 μM of the tesB gene product to theassay buffer containing the pimeloyl-CoA and incubating at 37° C. for 20min. The release of Coenzyme A was monitored by absorbance at 412 nm.The absorbance associated with the substrate only control, whichcontained boiled enzyme, was subtracted from the active enzyme assayabsorbance and compared to the empty vector control. The gene product oftesB accepted pimeloyl-CoA as substrate as confirmed via relativespectrophotometry (see FIG. 8) and synthesized pimelate as a reactionproduct.

EXAMPLE 2 Enzyme Activity of ω-transaminase Using Pimelate Semialdehydeas Substrate and Forming 7-Aminoheptanoate

A sequence encoding an N-terminal His-tag was added to the genes fromChromobacterium violaceum, Pseudomonas syringae, Rhodobactersphaeroides, and Vibrio Fluvialis encoding the ω-transaminases of SEQ IDNOs: 8, 10, 11 and 13, respectively (see FIGS. 7F and 7G) such thatN-terminal HIS tagged ω-transaminases could be produced. Each of theresulting modified genes was cloned into a pET21a expression vectorunder control of the T7 promoter and each expression vector wastransformed into a BL21[DE3] E. coli host. The resulting recombinant E.coli strains were cultivated at 37° C. in a 250 mL shake flask culturecontaining 50 mL LB media and antibiotic selection pressure, withshaking at 230 rpm. Each culture was induced overnight at 16° C. using 1mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanoateto pimelate semialdehyde) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanoate,10 mM pyruvate and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanoate and incubated at 25° C. for 4 h,with shaking at 250 rpm. The formation of L-alanine from pyruvate wasquantified via RP-HPLC.

Each enzyme only control without 7-aminoheptanoate demonstrated low baseline conversion of pyruvate to L-alanine See FIG. 14. The gene productof SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 accepted7-aminoheptanote as substrate as confirmed against the empty vectorcontrol. See FIG. 15.

Enzyme activity in the forward direction (i.e., pimelate semialdehyde to7-aminoheptanoate) was confirmed for the transaminases of SEQ ID NO 10,SEQ ID NO 11 and SEQ ID NO 13. Enzyme activity assays were performed ina buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM pimelate semialdehyde, 10 mM L-alanine and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase geneproduct or the empty vector control to the assay buffer containing thepimelate semialdehyde and incubated at 25° C. for 4 h, with shaking at250 rpm. The formation of pyruvate was quantified via RP-HPLC.

The gene product of SEQ ID NO 10, SEQ ID NO 11 and SEQ ID NO 13 acceptedpimelate semialdehyde as substrate as confirmed against the empty vectorcontrol. See FIG. 16. The reversibility of the ω-transaminase activitywas confirmed, demonstrating that the ω-transaminases of SEQ ID NO 8,SEQ ID NO 10, SEQ ID NO 11, and SEQ ID NO 13 accepted pimelatesemialdehyde as substrate and synthesized 7-aminoheptanoate as areaction product.

EXAMPLE 3 Enzyme Activity of Carboxylate Reductase Using Pimelate asSubstrate and Forming Pimelate Semialdehyde

A sequence encoding a HIS-tag was added to the genes from Segniliparusrugosus and Segniliparus rotundus that encode the carboxylate reductasesof SEQ ID NOs: 4 and 7, respectively (see FIGS. 7C and 7F), such thatN-terminal HIS tagged carboxylate reductases could be produced. Each ofthe modified genes was cloned into a pET Duet expression vector alongwith a sfp gene encoding a HIS-tagged phosphopantetheine transferasefrom Bacillus subtilis, both under the T7 promoter. Each expressionvector was transformed into a BL21[DE3] E. coli host and the resultingrecombinant E. coli strains were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication,and the cell debris was separated from the supernatant viacentrifugation. The carboxylate reductases and phosphopantetheinetransferases were purified from the supernatant using Ni-affinitychromatography, diluted 10-fold into 50 mM HEPES buffer (pH=7.5), andconcentrated via ultrafiltration.

Enzyme activity assays (i.e., from pimelate to pimelate semialdehyde)were performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM pimelate, 10 mMMgCl₂, 1 mM ATP and 1 mM NADPH. Each enzyme activity assay reaction wasinitiated by adding purified carboxylate reductase andphosphopantetheine transferase gene products or the empty vector controlto the assay buffer containing the pimelate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without pimelatedemonstrated low base line consumption of NADPH. See FIG. 9.

The gene products of SEQ ID NO 4 and SEQ ID NO 7, enhanced by the geneproduct of sfp, accepted pimelate as substrate, as confirmed against theempty vector control (see FIG. 10), and synthesized pimelatesemialdehyde.

EXAMPLE 4 Enzyme Activity of Carboxylate Reductase Using7-Hydroxyheptanoate as Substrate and Forming 7-Hydroxyheptanal

A sequence encoding a His-tag was added to the genes from Mycobacteriummarinum, Mycobacterium smegmatis, Segniliparus rugosus, Mycobacteriumsmegmatis, Mycobacterium massiliense, and Segniliparus rotundus thatencode the carboxylate reductases of SEQ ID NOs: 2-7—respectively (seeFIGS. 7A-7F) such that N-terminal HIS tagged carboxylate reductasescould be produced. Each of the modified genes was cloned into a pET Duetexpression vector alongside a sfp gene encoding a His-taggedphosphopantetheine transferase from Bacillus subtilis, both undercontrol of the T7 promoter.

Each expression vector was transformed into a BL21[DE3] E. coli host andthe resulting recombinant E. coli strains were cultivated at 37° C. in a250 mL shake flask culture containing 50 mL LB media and antibioticselection pressure, with shaking at 230 rpm. Each culture was inducedovernight at 37° C. using an auto-induction media.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugation.The carboxylate reductases and phosphopantetheine transferase werepurified from the supernatant using Ni-affinity chromatography, diluted10-fold into 50 mM HEPES buffer (pH=7.5) and concentrated viaultrafiltration.

Enzyme activity (i.e., 7-hydroxyheptanoate to 7-hydroxyheptanal) assayswere performed in triplicate in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 2 mM 7-hydroxyheptanal, 10mM MgCl₂, 1 mM ATP, and 1 mM NADPH. Each enzyme activity assay reactionwas initiated by adding purified carboxylate reductase andphosphopantetheine transferase or the empty vector control to the assaybuffer containing the 7-hydroxyheptanoate and then incubated at roomtemperature for 20 min. The consumption of NADPH was monitored byabsorbance at 340 nm. Each enzyme only control without7-hydroxyheptanoate demonstrated low base line consumption of NADPH. SeeFIG. 9.

The gene products of SEQ ID NO 2-7, enhanced by the gene product of sfp,accepted 7-hydroxyheptanoate as substrate as confirmed against the emptyvector control (see FIG. 11), and synthesized 7-hydroxyheptanal.

EXAMPLE 5 Enzyme Activity of ω-Transaminase for 7-Aminoheptanol, Forming7-Oxoheptanol

A nucleotide sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas syringae and Rhodobactersphaeroides genes encoding the ω-transaminases of SEQ ID NOs: 8, 10 and11, respectively (see FIGS. 7F and 7G) such that N-terminal HIS taggedω-transaminases could be produced. The modified genes were cloned into apET21a expression vector under the T7 promoter. Each expression vectorwas transformed into a BL21[DE3] E. coli host. Each resultingrecombinant E. coli strain were cultivated at 37° C. in a 250 mL shakeflask culture containing 50 mL LB media and antibiotic selectionpressure, with shaking at 230 rpm. Each culture was induced overnight at16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e., 7-aminoheptanolto 7-oxoheptanol) were performed in a buffer composed of a finalconcentration of 50 mM HEPES buffer (pH=7.5), 10 mM 7-aminoheptanol, 10mM pyruvate, and 100 μM pyridoxyl 5′ phosphate. Each enzyme activityassay reaction was initiated by adding cell free extract of theω-transaminase gene product or the empty vector control to the assaybuffer containing the 7-aminoheptanol and then incubated at 25° C. for 4h, with shaking at 250 rpm. The formation of L-alanine was quantifiedvia RP-HPLC.

Each enzyme only control without 7-aminoheptanol had low base lineconversion of pyruvate to L-alanine See FIG. 14.

The gene products of SEQ ID NO 8, 10 & 11 accepted 7-aminoheptanol assubstrate as confirmed against the empty vector control (see FIG. 19)and synthesized 7-oxoheptanol as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID 8, 10 & 11 accept7-oxoheptanol as substrate and form 7-aminoheptanol.

EXAMPLE 6 Enzyme Activity of ω-Transaminase Using Heptamethylenediamineas Substrate and Forming 7-Aminoheptanal

A sequence encoding an N-terminal His-tag was added to theChromobacterium violaceum, Pseudomonas aeruginosa, Pseudomonas syringae,Rhodobacter sphaeroides, Escherichia coli, and Vibrio fluvialis genesencoding the ω-transaminases of SEQ ID NOs: 8-13, respectively (seeFIGS. 7F and 7G) such that N-terminal HIS tagged ω-transaminases couldbe produced. The modified genes were cloned into a pET21a expressionvector under the T7 promoter. Each expression vector was transformedinto a BL21[DE3] E. coli host. Each resulting recombinant E. coli strainwere cultivated at 37° C. in a 250 mL shake flask culture containing 50mL LB media and antibiotic selection pressure, with shaking at 230 rpm.Each culture was induced overnight at 16° C. using 1 mM IPTG.

The pellet from each induced shake flask culture was harvested viacentrifugation. Each pellet was resuspended and lysed via sonication.The cell debris was separated from the supernatant via centrifugationand the cell free extract was used immediately in enzyme activityassays.

Enzyme activity assays in the reverse direction (i.e.,heptamethylenediamine to 7-aminoheptanal) were performed in a buffercomposed of a final concentration of 50 mM HEPES buffer (pH=7.5), 10 mMheptamethylenediamine, 10 mM pyruvate, and 100 μM pyridoxyl 5′phosphate. Each enzyme activity assay reaction was initiated by addingcell free extract of the ω-transaminase gene product or the empty vectorcontrol to the assay buffer containing the heptamethylenediamine andthen incubated at 25° C. for 4 h, with shaking at 250 rpm. The formationof L-alanine was quantified via RP-HPLC.

Each enzyme only control without heptamethylenediamine had low base lineconversion of pyruvate to L-alanine See FIG. 14.

The gene products of SEQ ID NO 8-13 accepted heptamethylenediamine assubstrate as confirmed against the empty vector control (see FIG. 17)and synthesized 7-aminoheptanal as reaction product. Given thereversibility of the ω-transaminase activity (see Example 2), it can beconcluded that the gene products of SEQ ID 8-13 accept 7-aminoheptanalas substrate and form heptamethylenediamine.

EXAMPLE 7 Enzyme Activity of Carboxylate Reductase forN7-Acetyl-7-Aminoheptanoate, Forming N7-Acetyl-7-Aminoheptanal

The activity of each of the N-terminal His-tagged carboxylate reductasesof SEQ ID NOs: 3, 6, and 7 (see Examples 4, and FIGS. 7B, 7E, and 7F)for converting N7-acetyl-7-aminoheptanoate to N7-acetyl-7-aminoheptanalwas assayed in triplicate in a buffer composed of a final concentrationof 50 mM HEPES buffer (pH=7.5), 2 mM N7-acetyl-7-aminoheptanoate, 10 mMMgCl₂, 1 mM ATP, and 1 mM NADPH. The assays were initiated by addingpurified carboxylate reductase and phosphopantetheine transferase or theempty vector control to the assay buffer containing theN7-acetyl-7-aminoheptanoate then incubated at room temperature for 20min. The consumption of NADPH was monitored by absorbance at 340 nm.Each enzyme only control without N7-acetyl-7-aminoheptanoatedemonstrated low base line consumption of NADPH. See FIG. 9.

The gene products of SEQ ID NO 3, 6, and 7, enhanced by the gene productof sfp, accepted N7-acetyl-7-aminoheptanoate as substrate as confirmedagainst the empty vector control (see FIG. 12), and synthesizedN7-acetyl-7-aminoheptanal.

EXAMPLE 8 Enzyme Activity of ω-Transaminase UsingN7-Acetyl-1,7-Diaminoheptane, and Forming N7-Acetyl-7-Aminoheptanal

The activity of the N-terminal His-tagged ω-transaminases of SEQ ID NOs:8-13 (see Example 6, and FIGS. 7F and 7G) for convertingN7-acetyl-1,7-diaminoheptane to N7-acetyl-7-aminoheptanal was assayedusing a buffer composed of a final concentration of 50 mM HEPES buffer(pH=7.5), 10 mM N7-acetyl-1,7-diaminoheptane, 10 mM pyruvate and 100 μMpyridoxyl 5′ phosphate. Each enzyme activity assay reaction wasinitiated by adding a cell free extract of the ω-transaminase or theempty vector control to the assay buffer containing theN7-acetyl-1,7-diaminoheptane then incubated at 25° C. for 4 h, withshaking at 250 rpm. The formation of L-alanine was quantified viaRP-HPLC.

Each enzyme only control without N7-acetyl-1,7-diaminoheptanedemonstrated low base line conversion of pyruvate to L-alanine See FIG.14.

The gene product of SEQ ID NOs: 8-13 acceptedN7-acetyl-1,7-diaminoheptane as substrate as confirmed against the emptyvector control (see FIG. 18) and synthesized N7-acetyl-7-aminoheptanalas reaction product.

Given the reversibility of the ω-transaminase activity (see example 2),the gene products of SEQ ID NOs: 8-13 accept N7-acetyl-7-aminoheptanalas substrate forming N7-acetyl-1,7-diaminoheptane.

EXAMPLE 9 Enzyme Activity of Carboxylate Reductase Using PimelateSemialdehyde as Substrate and Forming Heptanedial

The N-terminal His-tagged carboxylate reductase of SEQ ID NO 7 (seeExample 4 and FIG. 7F) was assayed using pimelate semialdehyde assubstrate. The enzyme activity assay was performed in triplicate in abuffer composed of a final concentration of 50 mM HEPES buffer (pH=7.5),2 mM pimelate semialdehyde, 10 mM MgCl₂, 1 mM ATP and 1 mM NADPH. Theenzyme activity assay reaction was initiated by adding purifiedcarboxylate reductase and phosphopantetheine transferase or the emptyvector control to the assay buffer containing the pimelate semialdehydeand then incubated at room temperature for 20 min. The consumption ofNADPH was monitored by absorbance at 340 nm. The enzyme only controlwithout pimelate semialdehyde demonstrated low base line consumption ofNADPH. See FIG. 9. The gene product of SEQ ID NO 7, enhanced by the geneproduct of sfp, accepted pimelate semialdehyde as substrate as confirmedagainst the empty vector control (see FIG. 13) and synthesizedheptanedial.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method for biosynthesizing a product selectedfrom the group consisting of pimelic acid, 7-aminoheptanoic acid,7-hydroxyheptanoic acid, heptamethylenediamine, and 1,7-heptanediol,said method comprising: (i) contacting 2-oxoglutarate with enzymescomprising a homocitrate synthase classified under EC 2.3.3.14 or EC2.3.3.13, a homocitrate dehydratase and a homoaconitate hydrataseclassified under EC 4.2.1.114, EC 4.2.1.36 or EC 4.2.1.33 aniso(homo)_(n)citrate dehydrogenase classified under EC 1.1.1.85, EC1.1.1.87, or EC 1.1.1.286, an indolepyruvate decarboxylase classifiedunder EC 4.1.1.43 or EC 4.1.1.74, a glutarate semialdehyde dehydrogenaseclassified under EC 1.2.1.20, 1.2.1.16, or EC 1.2.1.79, and aglutarate:CoA ligase classified under EC 6.2.1.6 to produceglutaryl-CoA, or contacting acetyl-CoA with enzymes comprising aβ-ketothiolase classified under EC 2.3.1.9, an acetyl-CoA carboxylaseclassified under EC 6.4.1.2, an acetoacetyl-CoA synthase classifiedunder EC 2.3.1.194, a 3-hydroxyacyl-CoA dehydrogenase classified underEC 1.1.1.-, an enoyl-CoA reductase classified under 4.2.1.-, aglutaconyl-CoA decarboxylase classified under EC 4.1.1.70, and anenoyl-[acp] reductase classified under EC 1.3.1.9 or a trans-2-enoyl-CoAreductase classified under EC 1.3.1.44 or a glutaryl-CoA dehydrogenaseto produce glutaryl-CoA; (ii) contacting said glutaryl-CoA with enzymescomprising a β-ketothiolase classified under EC 2.3.1.- or aβ-ketoacyl-[acp] synthase classified under EC 2.3.1.41, EC 2.3.1.179, EC2.3.1.180, a 3-hydroxyadipyl-CoA dehydrogenase classified under EC1.1.1.157, an enoyl-CoA hydratase, and an enoyl-[acp] reductaseclassified under EC 1.3.1.9 or a trans-2-enoyl-CoA reductase classifiedunder EC 1.3.1.44 to produce pimeloyl-CoA, or contacting saidglutaryl-CoA with enzymes comprising a β-ketothiolase classified underEC 2.3.1.- or a β-ketoacyl-[acp] synthase classified under EC 2.3.1.41,EC 2.3.1.179, EC 2.3.1.180, a 6-oxo-cyclohex-1-ene-carbonyl-CoAhydrolase classified under EC 3.7.1.-, a6-hydroxycyclohex-1-ene-1-carboxyl-CoA dehydrogenase classified under EC1.1.1.-, a cyclohexa-1,5-dienecarbonyl-CoA hydratase classified under EC4.2.1.100, a cyclohex-1-ene-1-carboxyl-CoA dehydrogenase classifiedunder EC 1.3.8.-, a cyclohex-1-ene-1-carboxyl-CoA hydratase classifiedunder EC 4.2.1-, a 2-hydroxycyclohexanecarboxyl-CoA dehydrogenaseclassified under EC 1.1.1.-, and a 2-ketocyclohexanecarboxyl-CoAhydrolase classified under EC 4.1.3.36 to produce pimeloyl-CoA; (iii)contacting said pimeloyl-CoA with enzymes comprising a thioesteraseclassified under EC 3.1.2., an aldehyde dehydrogenase classified underEC 1.2.1.3, a 6-oxohexanoate dehydrogenase classified under EC 1.2.1.63,a 7-oxoheptanoate dehydrogenase classified under EC 1.2.1.-, aglutaconate-CoA transferase classified under EC 2.8.3.12, or areversible CoA-ligase classified under EC 6.2.1.5 to produce pimelicacid or 7-hydroxyheptanoate, and/or contacting said pimeloyl-CoA withenzymes comprising an ω-transaminase classified under EC 2.6.1.- or adeacetylase classified under 3.5.1.62 to produce 7-aminoheptanoate orheptamethylenediamine; and (iv) optionally, contacting said7-hydroxyheptanoate from step (iii) with an alcohol dehydrogenaseclassified under EC 1.1.1.- to produce 1,7-heptanediol; wherein at leastone of said enzymes is a heterologous enzyme.
 2. The method of claim 1,wherein said thioesterase comprises an amino acid sequence that is atleast 90% identical to the amino acid sequence set forth in SEQ ID NO: 1and wherein said ω-transaminase comprises an amino acid sequence that isat least 90% identical to the amino acid sequences set forth in SEQ IDNOs: 8-13.
 3. The method of claim 1, further comprising contacting saidpimelic acid with a carboxylate reductase to form a terminal aldehydegroup as an intermediate in forming said 7-aminoheptanoate.
 4. Themethod of claim 3, wherein said carboxylate reductase comprises an aminoacid sequence that is at least 90% identical to the amino acid sequencesset forth in SEQ ID NOs: 2-7.
 5. The method of claim 1, wherein saidmethod is performed in a recombinant host cell by fermentation of saidrecombinant host cell.
 6. The method of claim 5, wherein saidfermentation comprises a principal carbon source obtained frombiological or non-biological feedstocks.
 7. The method of claim 6,wherein said biological feedstock is, or obtained from, monosaccharides,disaccharides, lignocellulose, hemicellulose, cellulose, lignin,levulinic acid, formic acid, triglycerides, glycerol, fatty acids,agricultural waste, condensed distillers' solubles, or municipal waste;or wherein said non-biological feedstock is, or obtained from, naturalgas, syngas, CO₂/H₂, methanol, ethanol, benzoate, or terephthalicacid/isophthalic acid mixture waste streams.
 8. The method of claim 5,wherein said recombinant host cell is a prokaryote.
 9. The method ofclaim 8, wherein said prokaryote is a genus selected from the groupconsisting of Escherichia, Clostridia, Corynebacteria Cupriavidus,Pseudomonas, Delftia, Bacillus, Lactobacillus, Lactococcus, andRhodococcus.
 10. The method of claim 5, wherein said recombinant hostcell is a eukaryote.
 11. The method of claim 10, wherein said eukaryoteis a genus selected from the group consisting of AspergillusSaccharomyces, Pichia, Yarrowia, Issatchenkia, Debaryomyces, Arxula, andKluyveromyces.
 12. The method of claim 5, wherein said recombinant hostcell comprises one or more of the following attenuated enzymes: alactate dehydrogenase, a menaquinol-fumarate oxidoreductase, an alcoholdehydrogenase producing ethanol, a pyruvate decarboxylase, a 2-oxoaciddecarboxylase generating isobutanol, a formate dehydrogenase, a polymersynthase, a NADPH-specific L-glutamate dehydrogenase, a NADP H-consumingtranshydrogenase, a pimeloyl-CoA dehydrogenase; an acyl-CoAdehydrogenase that degrades C7 building blocks and their precursors; aglutaryl-CoA dehydrogenase; or a pimeloyl-CoA synthetase; and/or whereinsaid recombinant host cell overexpresses one or more genes encoding: aPEP carboxykinase, a PEP carboxylase, a pyruvate carboxylase, a PEPsynthase, a L-alanine dehydrogenase; a NADH-specific L-glutamatedehydrogenase; a L-glutamine synthetase; a diamine transporter; adicarboxylate transporter; and/or a multidrug transporter.
 13. Themethod of claim 8, wherein said prokaryote is selected from the groupconsisting of Escherichia coli, Clostridium ljungdhalii, Clostridiumkluyveri, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidusmetallidurans, Pseudomaonas fluorescens, Pseudomonas putida, Pseudomonasoleavorans, Delftia acidovorans, Bacillus subtilis, Lactobacillusdelbrueckii, Lactobacillus lactis, and Rhodococcus equi.
 14. The methodof claim 10, wherein said eukaryote is selected from the groupconsisting of Aspergillus niger, Saccharomyces cerevisiae, Pichiapastoris, Yarrowia lipolytica, Issathenkia orientalis, Debaryomyceshansenii, Arxula adenoinivorans, and Kluyveromyces lactis.